Based on detailed investigation of the reaction mechanism with several Tz, revealing up to 96 % elimination after 2 hours, we have developed a TCO‐caged prodrug with 750‐fold reduced cytotoxicity compared to the parent drug and achieved in situ activation upon Tz/TCO click‐to‐release.Bond‐cleavage reactions triggered by bioorthogonal tetrazine ligation have emerged as strategies to chemically control the function of (bio)molecules and achieve activation of prodrugs in living systems. While most of these approaches make use of caged amines, current methods for the release of phenols are limited by unfavorable reaction kinetics or insufficient stability of the Tz‐responsive reactants. To address this issue, we have implemented a self‐immolative linker that enables the connection of cleavable  Bioorthogonal activation of caged phenols enables strategies to control the function of (bio)molecules in biological environments. While previous methods have been limited by slow click kinetics or insufficient stability, we show that introduction of a self‐immolative linker affords stable trans‐cyclooctene conjugates, as demonstrated by the design of a prodrug that upon bioorthogonal reaction with tetrazines leads to >200‐fold cytotoxicity. Biocompatible bond‐cleavage reactions have expanded the repertoire of bioorthogonal tools substantially, providing methods beyond chemical ligation to control the function of (bio)molecules in biological environments and living systems.trans‐cyclooctenes (TCO) triggered by click reaction with 1,2,4,5‐tetrazines (Tz) has gained significant attention due to exceptional reaction kinetics and molecular versatility.et al., TCO modified with a carbamate moiety in an allylic position  undergoes a 1,4‐elimination upon inverse electron demand Diels‐Alder (IEDDA) reaction with Tz and subsequent post‐click tautomerization, ultimately leading to the click‐triggered release of an amine or amino‐functionalized molecule (click‐to‐release).2) of less than 0.001 M−1 s−1 have been observed for the IEDDA reaction of vinyl ethers and Tz.et al. have developed rTCO‐ethers and rTCO‐carbonates that react substantially faster with Tz (k2>10 M−1 s−1), leading to click‐triggered release of alcohols and phenols .The bioorthogonal elimination of N,N’‐dimethylethylenediamine (DMEDA) can be used as a minimal SIL to achieve bioorthogonal elimination of phenols  that enables the use of carbamate linkages instead of ethers and carbonates. Here, we show that s Figure c. DependN‐methyl substituent with other functionalities moreover allows further modification, as very recently applied for the development of an rTCO‐caged fluorogenic resorufin derivative.DMEDA is commonly used as a self‐immolative linker or spacer in the design of cleavable and/or activatable compounds such as drug conjugates.p‐nitrophenyl (PNP) carbonate rTCO‐PNP (1) was treated with an excess of DMEDA and (following evaporation to afford crude intermediate 2) reacted with the PNP‐activated tyrosine‐BODIPY conjugate 3 to obtain the rTCO‐DMEDA‐caged compound 4 in an overall yield of 68 %. Saponification and subsequent PEGylation of the intermediate acid 5 via HBTU‐mediated amide coupling yielded rTCO‐DMEDA‐Tyr‐BODIPY (6) with a yield of 76 % over 2 steps  fluorophore that allows detection and relative quantification of all intermediates and products by HPLC using its characteristic absorption at a wavelength of 500 nm.6 triggered by reaction with Tz was then studied by HPLC‐MS. According to the current mechanistic understanding,A that can tautomerize to the releasing 1,4‐DHP isomer B1 and the non‐releasing 2,5‐DHP isomer B2 ,[2 (8),9)6  of <100 M−1s−1 have been reported for the click reaction of rTCO with DMT (7) and PA2 (8), pyrimidyl‐alkyl‐Tz such as 9 are known to be substantially more reactive, reaching k2 of >1000 M−1 s−1 (see Supporting Information for k2 determined in PBS at 37 °C).[Click‐to‐release of 2 Figure a. The N‐x Figure a. Based igure 7),7, 14 6 Figure b. While pseudo‐release’ that impedes accurate reaction monitoring.6 with Tz 7 and 8 in PBS at 37 °C. Despite a tailing peak for the SIL‐intermediate S, all reactants, tautomers, and products could be separated and identified , the non‐releasing tautomer B2, and the SIL‐intermediate S (as the product of TCO‐release) that upon self‐immolation afforded phenol P with an overall yield of 89 % after a reaction time of approx. 8 hours. In agreement with our previous findings,B1 was found due to rapid 1,4‐elimination of this labile intermediate. The respective pyridazine dead‐end Ox was identified as the only non‐releasing byproduct  leading to ‘2 (8) is the only Tz reported so far that allows efficient release (>90 %) of primary amines.A and shift the equilibrium to B1/B2, as shown for the reaction of 6 and 8  in Figure A was not detectable. Enhanced tautomerization leads to formation of instantaneously releasing B1 and slowly oxidizing B2, which can still tautomerize to B1 (via A), thereby enabling efficient release. In agreement with these mechanistic considerations, we observed 92 % release of the uncaged phenol P and 8 % of the respective oxidized dead‐end   and rTCO leads to formation of two initial click products  is either on the same (head‐to‐head) or the opposite side (head‐to‐tail) of the leaving group in allylic position of the TCO [3+ moiety) formed upon reaction of 6 with PymK (9) without provoking any pseudo‐release (for details see the Supporting Information). As expected, we observed immediate post‐click tautomerization (no A detected) and 1,4‐elimination (no B1 detected) leading to formation of the SIL‐intermediate S and stable B2 in a ratio of 7/3, which confirms favored click orientation with NH3+ in head‐to‐head position , revealing a first‐order rate constant (k1) of 1.0 h−1 for the cyclization of the DMEDA‐carbamate and a respective half‐life (t1/2) of 42 min .In contrast to the symmetric tetrazines s Figure a. DependO Figure b. Simila2 Figure a. Ammonie Figure c. As a rn Figure d. The unn Figure d, which CA4, Figure CA4 have motivated the development of several therapeutic strategies, in particular by applying prodrugs such as CA4‐phosphate  to overcome the poor water‐solubility of the parent drug.To demonstrate application of the Tz/TCO‐DMEDA strategy for the bioorthogonal release of phenols, we designed a prodrug of the anti‐mitotic and anti‐angiogenic agent combretastatin A‐4 (CA4 was enabled by PNP‐activation of the free phenolic OH‐group yielding PNP‐CA4 (10)  was first treated with an excess of DMEDA and then reacted with PNP‐CA4 (10). Saponification of the methyl ester followed by HBTU‐mediated coupling of the intermediate acid with 2‐aminoethanesulfonic acid (taurine) afforded sulfo‐cTCO‐DMEDA‐CA4 (12) in an overall yield of 34 %  Figure a. To fac2 Figure b. To inc% Figure b.12 by reaction with tetrazines 7 and 9 was monitored by HPLC‐MS as described above. The relative abundance of all intermediates and products was calculated based on the UV absorbance at 254 nm and confirmed via quantification of released CA4 by external calibration (see Supporting Information). In agreement with data previously reported,12 with DMT (7) resulted in 96 % release of CA4 already after 2 hours, reaching an endpoint of 98 % CA4 and 2 % of the respective oxidized dead‐end after a total reaction time of approx. 4 hours  did not lead to favored formation of the initial click product with the NH3+‐substituent (K) in head‐to‐head position, as indicated by an overall CA4 release of 50 %. Accordingly, 50 % of non‐releasing stable B2 with K in head‐to‐tail position was detected , which we hypothesize is due to CA4 being a better leaving group than the tyrosine‐BODIPY conjugate P  was needed to reduce the cell count by 50 % (IC50) over the course of a 72 h treatment . In situ reaction of 12 with DMT (7) and PymK (9) at a non‐toxic Tz concentration of 5 μM restored drug cytotoxicity via Tz‐triggered bioorthogonal release of CA4, shifting the IC50 from 900 nM to 4.1 nM (220‐fold) and 6.4 nM (140‐fold), respectively  alone, only very low signal was detected upon bioorthogonal release of CA4 via in situ reaction of both compounds , matching the CA4 control and its expected impact on tubulin polymerization.Based on these results we investigated the cytotoxicity of prodrug t Figure d, demons2H, NH3+) and their effect on post‐click tautomerization and 1,4‐elimination. We achieved >95 % release of the phenolic drug CA4 upon Tz‐triggered cleavage of prodrug 12 in PBS within 2 hours. Notably, we have observed a 750‐fold lower cytotoxicity of the prodrug compared to CA4, also demonstrating high stability of TCO‐DMEDA‐phenol conjugates. In situ reaction of 12 with Tz confirmed efficient bioorthogonal release of CA4 resulting in a substantially (up to 220‐fold) increased cytotoxicity. Based on these results, we aim to combine TCO‐DMEDA‐caging with next‐level chemical tools for bioorthogonal bond‐cleavage to control post‐click tautomerization and elimination, thereby accelerating the Tz/TCO click reaction while achieving fast and complete release of phenolic drugs.In summary, we show that DMEDA can be applied as a minimal self‐immolative linker for the design of bis(carbamate)‐linked TCO‐caged phenols that can be cleaved and thereby activated by bioorthogonal reaction with tetrazines. The mechanism of the overall bond‐cleavage process upon IEDDA‐ligation with selected symmetrical and unsymmetrical Tz was investigated in detail, with emphasis on Tz‐substituents modified with directing groups  for preparative RP‐HPLC or a Luna 10 μm Silica (2) 100 Å, LC Column 250×21.2 mm (Phenomenex) for preparative NP‐HPLC purifications. Silica gel 60 (40‐63 μm) was purchased from Merck. HPLC‐MS (LCMS) analysis was performed on a Nexera X2 system (Shimadzu) comprised of LC‐30AD pumps, a SIL‐30AC autosampler, a CTO‐20AC column oven, and a DGU‐20 A5/3 degasser module. Detection was done using an SPD−M20 A photo diode array, an RF‐20Axs fluorescence detector, an ELS‐2041 detector (JASCO) and an LCMS‐2020 mass spectrometer (ESI). All separations were performed using a Waters XSelect CSH C18 2.5 μm (3.0×50 mm) column XP at 40 °C and a flowrate of 1.7 mL/min. Used gradients : Acidic HPLC conditions (acetonitrile/0.1 % formic acid): 0 min, 5 % – 0.15 min, 5 % – 2.20 min, 98 % – 2.50 min, 98 %; buffered HPLC conditions : 0 min, 5 % – 0.15 min, 5 % – 2.2 min, 98 % – 2.5 min; 98 %. 1H and 13C NMR spectra were recorded on a Bruker Ascend 600 MHz spectrometer at 20 °C. Chemical shifts (δ) are reported in ppm relative to tetramethylsilane and calibrated using solvent residual peaks. HRMS analysis of aqueous or acetonitrile solutions of the compounds (sample concentration: 10 ppm) was carried out on an Agilent 6545 Q‐TOF mass spectrometer equipped with an Agilent Dual AJS ESI source. The mass spectrometer was connected to a liquid chromatography system comprised of an Agilent G7167B multi sampler, an Agilent G7120 A binary pump with degasser and an Agilent G7116B oven (Agilent). A SecurityGuard Cartridge (Phenomenex) was used as a stationary phase. Data evaluation was performed using Agilent MassHunter Workstation Qualitative Analysis 10.0. Stopped‐flow measurements were performed using an SX20‐LED stopped‐flow spectrophotometer (Applied Photophysics) equipped with a 535 nm LED to monitor the characteristic tetrazine visible light absorbance (520‐540 nm).Synthesis: Detailed experimental procedures for the synthesis of compounds 2–6, 10–12 and further intermediates are provided in the Supporting Information, including compound characterization data .Click‐to‐release monitoring (HPLC): Click‐to‐release reactions were performed in HPLC vials at 37 °C using the temperature‐controlled HPLC autosampler . For acidic HPLC conditions, the aqueous solvent was prepared by addition of 2.5 mL of formic acid to 2.5 L of HPLC‐water to yield a final concentration of 0.1 % formic acid. For buffered HPLC conditions, the aqueous solvent was prepared by addition of 625 μL of 10 M ammonium formate  to 2.5 L of HPLC‐grade water followed by adjusting the pH to 8.4 by addition of 25 % aqueous ammonia . Since its pH declines over time, this volatile buffer was freshly prepared each day. HPLC‐grade acetonitrile was used without any additives. Stock solutions of rTCO‐DMEDA‐Tyr‐BODIPY (6) and sulfo‐cTCO‐DMEDA‐CA4 (12) were prepared at a concentration of 20 mM in DMSO. Tetrazine stock solutions of DMT (7), PA2 (8) and PymK (9) were prepared at a concentration of 10 mM in DMSO.6): The stock solution of 6 was diluted with PBS to a concentration of 100 μM in an LCMS vial . Tetrazine stock solutions were diluted with PBS to a concentration of 200 μM in an LCMS vial , and the click‐to‐release reaction was initiated by addition of the diluted TCO solution (125 μL) to obtain starting concentrations of 50 μM TCO and 100 μM Tz. The samples were immediately incubated at 37 °C in the autosampler and subjected to serial HPLC analysis. All measurements were conducted in triplicates.rTCO‐DMEDA‐Tyr‐BODIPY (12): The stock solution of 12 was diluted with DMSO  to give a 10 mM stock solution. Tz stock solution (10 μL) was added to PBS , and the click‐to‐release reaction was initiated by addition of the stock solution of 12 (5.24 μL) to obtain starting concentrations of 50 μM TCO and 100 μM Tz (in 10 % DMSO/PBS). The samples were immediately incubated at 37 °C in the autosampler and subjected to serial HPLC analysis. All measurements were conducted in triplicates.sulfo‐cTCO‐DMEDA‐CA4 : An approx. 20 mM stock solution of 4rTCO‐PEG7) (extinction coefficient 510 M−1 cm−1 at 520 nm), quantifying the decrease in tetrazine absorbance upon reaction with TCO. The initial DMSO stock was diluted with PBS to prepare solutions for stopped‐flow analysis at a final TCO concentration of 1 mM. Stock solutions (20 mM) of DMT (7), PA2 (8), and PymK (9) in DMSO were prepared. Serial dilution into PBS gave solutions for stopped‐flow analysis at a Tz concentration of 100 μM. The reagent syringes of the stopped‐flow spectrophotometer  were loaded with tetrazine and TCO solutions and the instrument was primed. Stopped‐flow measurements were done in sextuplicate for each Tz. Reactions were conducted at 37 °C and recorded automatically at the time of acquisition. Data sets were analyzed by fitting an exponential decay using Prism 6 (GraphPad) to calculate the observed pseudo‐first‐order rate constants that were converted into second‐order rate constants  by dividing through the TCO concentration.Cell viability assays: HT1080 human fibrosarcoma cells (ATCC) were cultivated in EMEM  supplemented with 10 % fetal bovine serum and 1 % antibiotic/antimycotic solution  at 37 °C and 5 % CO2. HT1080 cells were seeded into 96‐well plates (triplicates for each group) at 10.000 cells per well and allowed to grow overnight. The medium was removed and a dilution series of sulfo‐cTCO‐DMEDA‐CA4 (12) or the parent drug CA4 in growth medium  was added to the cells . For release experiments the same concentrations of 12 were used, while a stock solution of DMT (7) or PymK (9) was added to obtain final concentration of 5 μM of the respective tetrazine. Incubation was carried out for 72 h. Cell viability was assessed by replacing the medium with 100 μL of PrestoBlue solution  followed by incubation for 30 minutes at 37 °C. Read‐out of the fluorescence signal was carried out using a PerkinElmer EnSpire Multimode Plate Reader and data processing was done in GraphPad Prism. Following the same procedure, cells were treated with DMT (7), PymK (9), or 1,3‐dimethylimidazolidin‐2‐one (=byproduct of the self‐immolation process) with concentrations of up to 10 μM, revealing no significant effect on cell viability.Prodrug stability and cell imaging: Additional data and experimental details regarding (i) the stability of prodrug 12 in PBS and cell growth medium and (ii) confirmation of the mechanistic basis of cytotoxicity upon click activation of 12 via fluorescence microscopy after microtubules staining are included in the Supporting Information.The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Mandevilla Lindl. is an important genus of the Apocynaceae family, not only as ornamental plants but also for its medicinal uses. In Brazil, Mandevilla species are indicated to treat asthma and skin infections, their anti-inflammatory potential and wound healing properties are also reported in the literature. Concerning their chemical composition, this group of plants is a conspicuous producer of pregnane glycosides. Mandevilla dardanoi is an endemic species from the Brazilian semiarid region not studied by any phytochemical methods. In view of the medicinal potential of Mandevilla species, this study aimed to isolate new pregnane glycosides from M. dardanoi. To achieve this main goal, modern chromatography techniques were employed. Five new pregnane glycosides, dardanols A-E, were isolated from the roots of M. dardanoi by HPLC. Their structures were determined using extensive 1D and 2D-NMR and mass spectrometry (MSn and HRESIMS) data. The cytotoxicity and the anti-inflammatory potential of these compounds were evaluated. The first was evaluated by measuring proinflammatory cytokines and nitric oxide production by stimulated macrophages. Dardanols were able to inhibit the production of nitric oxide and reduce IL-1β and TNF-α. The current work demonstrates the chemodiversity of Brazilian semiarid species and contributes to amplifying knowledge about the biological potential of the Mandevilla genus. Mandevilla Lindl. is one of the largest neotropical genera of the Apocynaceae family, this group comprises approximately 170 species + , indicating 13 indices of hydrogen deficiency (IHD) (Compound cy (IHD) .1H and 13C NMR data  corresponding to an acetal group hydrogen, δH 5.41 , an olefinic hydrogen, δH 4.58  and 3.81 , oxymethinic hydrogens, 1.06  and 0. 94 , methyl hydrogens, associated with their respective carbons at δC 104.2 (C-16), 122.1 (C-6), 78.9 (C-20), 77.5 (C-3), 16.9 (C-18) and 19.7 (C-19), are in agreement with the values described for the aglycone illustrol, a seco-norpregnane derivative, isolated from M. illustris and reported only once 25D +18 . Its molecular formula was determined to be C50H78O17 by HRESIMS, with m/z 973.5086 [M + Na]+ , indicating 12 hydrogen deficiency indices. The 1H and 13C NMR data were similar to compound 1 being assigned to illustrol-type aglycone , 4.78 , 5.15 , and 5.40 , as well as the presence of methyl hydrogen signals at δH 1.51 , 1.34 , 1.39  and 1.28 , and of the hydrogens of the methoxy groups at δH 3.33 , δH 3.39 , δH 3.46  and δH 3.38 , indicated the existence of four osidic units in compound 2.Compound aglycone . When co1H and 13C NMR data of the osidic units also resembled compound 1, allowing us to establish 2 as illus-trol-3-O-β-L-diginopyranosyl-(1-4)-α-L-sarmentopyranosyl-(1-4)-β-D-cymaropyranosy-(1-4)-β-L-4 acetoxylsarmentopyranosyl, a new natural product named dardanol B.The 3 was isolated as a white powder with a positive optical rotation of [α]25D +13 . Its molecular formula was determined to be C58H94O21 by HRESIMS, with m/z 1109.6252 [M − H2O + H]+ , indicating 12 hydrogen deficiency indices. The NMR data resembled that of the aglycone illustrol; however, some differences were observed for the carbons at δC 38.9, 46 and 62.5, assigned to C-8, C-13 and C-17, respectively, with C-8 and C-17 being deprotected and C-13 protected when compared to the chemical shifts of these carbons in compound 1. These data were associated with one lower hydrogen deficiency index compared with compound 1, suggesting the opening of the C-14-O-C-16 epoxide. The signal at δH 6.88 (s), uncorrelated in the HSQC spectrum, was assigned to the OH located at C-14. This signal showed correlations in the HMBC spectrum with the signals at δC 46.0 and 110.1 that were assigned to C-13 and C-14, respectively. Additionally, in the HMBC spectrum, we observed correlations of the signal at δH 1.40 (3H-18) with the carbons at δC 110.1 (C-14) and in the HMBC spectrum at δC 46.0 (C-13) and with δC 62.5, which was assigned to C-17. The signal at δH 5.17, whose corresponding carbon in the HSQC spectrum was δC 105.8, was assigned to C-16. The signal at δH 5.17 correlated with δC 46.0 (C-13) and with δC 62.5 (C-17). A correlation of this signal with δC 71.4, assigned to C-21, was also observed. A correlation was also observed with the signal at δC 54.5 that was assigned to methoxy bound at position 16. The coupling constant of H-17  and a singlet for H-16 demonstrated near 90-degree angulation between these hydrogens, and thus, the aglycone was defined as shown in 1 data and high-resolution mass spectrometry confirmed the presence of five 1–4 bonded osidic units identical to compound 1 and inserted in C-3 -α-L-sarmentopyranosyl-(1-4)-β-D-cymaropyranosy-(1-4)-β-D-oleandropyranosyl-(1-4)-β-L-4-acetoxylsarmentopyranosyl, a new natural pro-duct named dardanol C.To reject the possibility that the aglycone had been formed in the process of separating the compounds, a direct infusion on the ESIMS/MS was performed with the crude ethanolic extract. The presence of the ion at mpound 3 . Thus, c4 was isolated as a white powder with a positive optical rotation of [α]25D +17 . Its molecular formula was determined to be C28H44O8 by HRESIMS, m/z 531,2923 [M + Na]+ , indicating 7 hydrogen deficiency indices. The NMR data for the aglycone of this compound were similar to compound 3, and it could be concluded that it was of the same type as OH at C-14 and OCH3 at C-16. In the 13C NMR spectrum, it was possible to observe six carbons at δC 99.0, 33.2, 79.4, 67.4, 71.4 and 20.4 in addition to a signal for methoxyl at δC 55.3 compared with the literature, and the other osidic units of compounds 1–3 were marked as diginopyranose. The H-1′ coupling constant at 11.6 and 4.0 Hz was compatible with β-glycosidic bonding, and the C-2′ value at δC 33.5 allowed the configuration of the osidic unit to be assigned as L-diginopyranosyl. The molecular mass of this compound was also found in the analysis of the crude extract by ESIMS/MS, which again rejected the possibility of artifacts. Thus, compound 4 was identified as seco-illustrol-3-O-β-L-diginopyranosyl, a new natural product named dardanol D.Compound 5 was isolated as a white powder with a positive optical rotation of [α]25D −19 . Its molecular formula was determined to be C58H92O21 by HRESIMS, with m/z 1147.6037 [M + Na]+ . The 1H and 13C NMR data showed signals characteristic of pregnane glycosides. The hydrogen signals at δH 5.00 , 5.89  and 4.55  together with carbons at δC 93.5, 109.7 and 74.7 are in agreement with the values described for the velutinol aglycon, reported only in M. velutinus 25D + 14 ; 1H and 13C NMR data, see m/z 1117.5918 [M + Na]+ .Dardanol B (2): white powder; [α]25D + 18 ; 1H and 13C NMR data, see m/z 973.5086 [M + Na]+ .Dardanol C (3): white powder; [α]25D + 13 ; 1H and 13C NMR data, see m/z 1109.6252 [M − H2O + H]+ .Dardanol D (4): white powder; [α]25D + 17 ; 1H and 13C NMR data, see m/z 531,2923 [M + Na]+ .Dardanol E (5): white powder; [α]25D − 19 ; 1H and 13C NMR data, see m/z 1147.6037 [M + Na]+ .Mandevilla dardanoi by modern chromatographic techniques and characterized by comprehensive spectroscopic data. Among them, compounds 3 and 4 contained a novel seco-pregnane-type aglycone. Dardanols A, B, C and E showed anti-inflammatory potential by inhibiting the production of nitric oxide and reducing the pro-inflammatory cytokines IL-1β anFive new pregnane steroidal glycosides (dardanols A–E) were isolated from the ethanolic extract of d TNF-α in stimulated macrophages. These findings enrich the knowledge of the chemodiversity and biological potential of Caatinga species.


Because of the well-defined steps in its progression (adenoma - Tis - T1 invasive cancer- T2 advanced cancer with metastases), colorectal cancer (CRC) represents a model for investigating the effects of angiogenesis throughout tumor development.  The aim of this study is to determine the role of VEGF expression by immunohistochemistry (IHC) in normal epithelium, dysplasia, carcinoma of colorectal specimens and to correlate the same with tumor grade, stage, nodal status, and metastasis.Introduction: Colorectal cancer is one of the malignancies in which angiogenesis has been implicated and its importance at different stages of malignant disease. Neovascularization begins when the angiogenic switch is turned on and when angiogenesis activators outweigh angiogenic inhibitors. The process of blood vessel formation is regulated through several growth factor systems. The vascular endothelial growth factor (VEGF) system is one of the important ones and may consequently be a key system in relation to different aspects of colorectal carcinoma treatment.Methods: This is a retrospective study on paraffin blocks of 50 colon cancer specimens, 40 adenoma specimens, and 10 normal colonic mucosa specimens. Immunohistochemical stain for VEGF was done on the sections along with controls. Monoclonal antibody detected against VEGF antigen was observed in the cytoplasm of the tumor cells and the intensity of VEGF expression in individual tumor cells was scored on a scale of 0 (no staining) to 3 (strong intensity), and the percentage of cells with VEGF staining at each intensity was estimated from 0 to 100. Pearson’s Chi-squared test and Mann-Whitney test were used to determine significant clinicopathological differences between VEGF expression in positive and negative tumors.Results: In normal epithelium, VEGF immunoreactivity was seen in all 10 cases with high intensity. Among adenomas, VEGF expression was seen in 26 (65%) of the 40 cases. Out of which in tubular adenomas VEGF expression was seen in 13 cases (60%) and negative in eight cases (40%). In tubulo villous adenoma, VEGF expression was seen in nine cases (60%) and negative in six cases (40%). Villous adenomas showed VEGF expression in all four cases (100%). In adenocarcinoma, VEGF expression was seen to be expressed in 42 cases (84%) and negative in eight cases (16%) and expression was higher in low-grade carcinomas (70%) compared to high-grade carcinomas. A significant difference in the expression of VEGF among adenomas and carcinomas was observed with higher intensity present in adenoma when compared to carcinoma.Conclusion: Expression of VEGF could be considered as an early carcinogenic factor in colorectal carcinomas as it is expressed in higher intensity in the precancerous lesion and low-grade and stage 1 adenocarcinoma. Hence, we infer that early colorectal carcinomas are an important model for targeted therapy with antiangiogenic factors for VEGF.  Colorectal cancer (CRC) is the third most common cancer occurring worldwide with an estimation of one million new cases diagnosed each year . In receThis is a retrospective study on 100 paraffin blocks of 50 colon cancer specimens, 40 adenoma specimens, and 10 normal colonic mucosa specimens received in the Department of Pathology at Sri Ramachandra University over a period of three years. Immunohistochemical stain for VEGF was done on the sections along with controls.Permission from the Institutional Ethics Committee was obtained prior to commencing the study (IEC NO: CSP-MED/13/JUN/07/34). Gross findings were recorded and clinical data were obtained from the medical record section and available local area computer network in service. Paraffin blocks made after routine processing of resected colectomy specimens were used for the same. Blocks with sections containing tumors, adenomas, and normal colonic mucosa were chosen for the immunohistochemistry (IHC) study. This IHC study was conducted on an estimated sample size of 50 cases of histopathology-proven colonic carcinoma, 40 adenomas, and 10 normal colonic mucosa.Hematoxylin and eosin (H&E) stained sections were used to grade colon cancer by architectural and cytological features. Other histopathological features observed were adjacent dysplasia and adenomas. Immunohistochemical staining for VEGF was performed on all 100 cases. Monoclonal antibody detected against VEGF antigen was observed as brown color in the cytoplasm of the tumor cells and the intensity of VEGF expression in individual tumor cells was scored on a scale of 0 (no staining) to 3 (strong intensity) . Pearson’s Chi-squared test and Mann-Whitney test were used to determine significant clinicopathological differences between VEGF expression in positive and negative tumors. Differences were considered statistically significant when p value was <0.05.We studied holoprosencephaly (HPE) proven cases of 50 colorectal carcinomas, 40 colonic adenomas which included 21 tubular adenomas, 15 tubulovillous adenomas, and 4 villous adenomas diagnosed in the Department of Pathology, Sri Ramachandra Medical College and Research Institute over a period of three years. We included 10 normal colonic tissues away from the lesion from the CRC specimens.In our study of colon cancers, the age of the patients ranged from 27 to 85 years. The highest incidence was noted in the fifth decade. In adenomas, the highest incidence was noted in the sixth decade of life. We found that colon cancer occurred predominantly in men as compared to women with a male-to-female ratio of 3.1 : 1 and in adenomas also males were affected predominantly than females with a ratio of 2.3:1. Rectum was the most common site involved in both colonic cancer as well as in adenomas . The size of the CRCs ranged from 2.5 to 12 cm out of which up to 5 cm was 48% and more than 5 cm was 52%. In adenomas size ranged from 0.2 to 8 cm out of which in tubular adenomas size ranged from 0.2 to 2 cm and villous adenoma was slightly bigger in size and ranged from 0.3 to 8 cm. There is no definite predilection for large tumors. In CRCs, grade 1 was categorized as well differentiated, grade 2 as moderately differentiated, and grade 3 as poorly differentiated. In this study grade 2 is more common (69%) followed by grade 1 (18%).In our study most of the tumors (50%) belong to pT3  of the TNM  classification, followed by pT2 - (44%) (tumor invades muscularis propria). Regional lymph node metastasis was seen in 48% with N1 - 36%  and N2 - 12% (metastasis in more than four lymph nodes). Nodal metastasis was absent in 52%. Clinically no distant metastasis (cM0) was noted in all the cases except for one case. When the comparison was made for stage, stages 1 and 3 predominated over stages 2 and 4. The high stage was noted predominantly in males and more common in the age group of >60 years.We analyzed the expression of VEGF in normal colonic epithelium, tubular adenoma, tubulovillous adenoma, villous adenoma, and carcinoma. We noted that the staining of VEGF immunohistochemical stain had taken up in the cytoplasm of allcases .In normal epithelium, VEGF immunoreactivity was seen in all 10 cases with a high intensity of 3+ Figure .Among adenomas, VEGF expression was seen in 26 (65%) of the 40 cases with an intensity of 2+ in 15 cases and 3+ in 11 cases. In tubular adenoma, 13 cases showed positivity with an intensity of 2+ in nine cases and 3+ in four cases . The H score ranged from 40 to 300 in villous adenomas and 70 to 300 in tubular adenomas Table .According to the age, VEGF was expressed high in the age group of 61-70 in all the adenomas and there is no significant difference in the expression of VEGF according to the site.In colorectal carcinomas, VEGF expression when compared based on the age of the patients, showed positivity in 18 cases (58.1%) among the age group <45 years and positivity in eight cases (42.1%) among patients >45 years of age. Thus there is no significant difference with VEGF expression and age of the patients (p = 0.273). When VEGF expression was compared to the size of the tumor, 11 cases (42.3%) were positive in tumors of size up to 5 cm and 15 cases (62.3%) were positive in tumors >5 cm with a p-value of 0.153 which was statistically insignificant.The clinicopathological data of VEGF expression is given in Table The VEGF expression was analyzed with grade and TNM staging in colorectal carcinomas; the data are summarized in Table In our study, expression of VEGF was seen positive in 7/10 (70%) cases in grade 1 with an intensity of 3+ and showed almost equal positivity in grades 2 and 3 with an intensity of 2+ (47% and 52%) (p=0.443) Figures -7.Expression of VEGF was also analyzed for pT staging, 13 cases of pT2 stage tumors were positive with an intensity of 3+ in eight cases and 2+ in five cases. Some 12 cases of pT3 were positive with an intensity of 2+ in eight cases and 3+ in four cases and out of three cases in pT4 only one case was positive which showed an intensity of 2+ (p=0.600) Figure .In nodal metastasis, VEGF was positive in eight (44.8%) cases/18 cases in N1 and three (50%)/six cases in N2 were positive with variable intensity and a p-value of 0.684, which was not significant. When the expression of VEGF and stage were compared, stage 1 showed maximum expression in 13 cases (65%), H scores ranging from 10 to 300, stage 2 showed expression in four cases 44.4%) H score ranging from 100 to 300, stage 3 (40%) in eight cases score ranging from 30 to 300, and stage 4 in one case (100%) with a score of 280 (p=0.303). In our study, the p-value was higher so there was no correlation with the grade and stage of the tumor  , moderately differentiated (grade II), and poorly differentiated (grade III). In this study grade 2 was more common (69%) followed by grade 1 (18%) which correlated with the study done by Yalcin et al. . In our In this study we studied the expression of VEGF in normal colonic epithelium, tubular adenoma, tubulovillous adenoma, villous adenoma, and carcinoma. In normal epithelium VEGF immunoreactivity was seen in all the 10 cases with a maximum intensity and a score of 300 which correlated with the study of Hanrahan et al. . In his In adenomas, VEGF expression was seen in 25 (64.1%) cases out of which VEGF expression was seen in 12 cases (60%) of tubular adenomas. The VEGF expression in tubulo villous adenoma was seen in nine cases (60%) and villous adenoma showed VEGF expression in all four cases (100%). There was a significant difference between the expression of VEGF in villous adenoma and tubular adenoma (p=0.05). The H score ranged from 40 to 300 in villous adenoma and 70 to 300 in tubular adenoma and in carcinomas expression was seen in 51% of cases.From our study there was a slight increase in the expression of VEGF in colonic adenomas than the carcinomas with a mean of 216 in adenoma and 195 in carcinoma whereas in the study conducted by Abdou et al. , the VEGMolecular studies on transgenic mouse models have shown that angiogenesis begins in the premalignant phase of tumorogenesis, when dysplastic lesions acquire an increased microvasculature . Thus thIn colorectal carcinomas, VEGF expression was seen in 18 cases (58.1%) <45 years of age and positive in eight cases (42.1%) >45 years. There was no significant difference in age with VEGF expression (p = 0.273). When VEGF expression was compared to the size, 11 cases (42.3%) were positive among tumors up to 5 cm and 15 cases (62.3%) were positive among tumors > 5 cm (p=0.153) which was statistically insignificant. Similar findings were observed by Abdou et al.  and BendTumor grade and stage in relation to VEGF positivity were analyzed for correlation. The VEGF was expressed high in grade 1 with a median score of 280 and a score of 210 in grade 2 and a median score of 180 in grade 3. Statistical data showed a p-value of 0.684. Conflicting studies were published considering VEGF expression and tumor differentiation. The VEGF expression was significantly more intense in poorly differentiated tumors in comparison with the well-differentiated ones in CRCs. In the present study, there was an inverse relationship between tumor grade and VEGF expression though it was insignificant. Similar findings were observed by Uner et al.  and AbdoIn our study when the expression of VEGF and stage was compared a low expression was seen in advanced stage tumor. Stage 1 showed a maximum expression of 13 cases (65%), stage 2 showed an expression in four cases (44.4%), stage 3 (40%) in eight cases, and stage 4 in one case with a p-value of 0.303. According to Zheng et al. , HanrahaGeorge et al.  and SaadIn view of the association of high VEGF expression in normal epithelium away from the tumor, adenomas, tumors of low grade, early stage, and negative nodal status by immune histochemical assessment, it could be suggested that VEGF may be an early carcinogenic factor in CRC. VEGF may be required for CRC development in early stages but as tumors become less differentiated, more aggressive, and deeply invading, it is no longer in need of VEGF that appears to be downregulated. So other angiogenic factors could have the upper hand in this stage of aggression and advancement of CRCs.From our study reduced expression of VEGF in tumors with high grade and advanced stage necessitates further correlation with the molecular studies in CRCs in this part of India.From the above findings of our study, we conclude that VEGF could be considered as an early carcinogenic factor in colorectal carcinomas as it is expressed in higher intensity in precancerous lesions like adenoma and low-grade and stage 1 adenocarcinomas. Hence we infer that early colorectal carcinoma is an important model for targeted therapy with antiangiogenic factors for VEGF. The decreased expression of VEGF in higher grades and stages of adenocarcinomas suggests that it has a reduced role in tumor progression. Hence we suggest that the reduced role of VEGF in tumors with high grade and advanced stage necessitates further study of the molecular pathology of CRC in this part of India.


The proliferation of Internet-of-Things has promoted a wide variety of emerging applications that require compact, lightweight, and low-cost optical spectrometers. While substantial progresses have been made in the miniaturization of spectrometers, most of them are with a major focus on the technical side but tend to feature a lower technology readiness level for manufacturability. More importantly, in spite of the advancement in miniaturized spectrometers, their performance and the metrics of real-life applications have seldomly been connected but are highly important. This review paper shows the market trend for chip-scale spectrometers and analyzes the key metrics that are required to adopt miniaturized spectrometers in real-life applications. Recent progress addressing the challenges of miniaturization of spectrometers is summarized, paying a special attention to the CMOS-compatible fabrication platform that shows a clear pathway to massive production. Insights for ways forward are also presented. Conventional high-performance spectrometers are typically based on bulky and costly systems with large dispersive components, long optical path length, and movable mechanisms. To meet the requirements of various application scenarios where the portability, cost, robustness, and power consumption are paramount metrics, such as portable or wearable sensing devices for healthcare, food safety monitoring5, smartphone-based spectrometers, drone-based remote sensing6 and space exploration8, substantial progresses have been made in miniaturizing spectrometers while maintaining adequate performance during the past decades. The projected market for mini- and micro- spectrometers has gone up to about 900 million dollars9, which stimulates considerable research efforts from both academia and industry. To name a few, Fraunhofer ENAS announced its goal of batch producing “one gram spectrometer” for smartphone integration at the cost of around one dollar10, whilst Rockley Photonics released a whitepaper aiming to realize high-yield manufacturable on-chip spectrometers for “clinic on the wrist” health monitoring11. Clearly, efforts are driven toward exploring miniaturized spectrometers that are mass-manufacturable for the consumer market.Optical spectrometer is one of the most essential instruments in numerous fields, including chemical engineering, materials analysis, astronomical science, medical diagnosis and biological sensing20. While some work has indeed advanced the state-of-the-art in miniaturized spectrometers, they may suffer from insufficient performance and low technology readiness levels in connection with commercialization. For instance, ultra-compact spectrometers at the scale of dozens of microns based on single compositionally engineered nanowires have been demonstrated, representing the smallest footprint on record14, which for sure, is recognized as a milestone for miniaturized spectrometers. However, a resolution of 5~10 nm is far away from being adequate for applications like glucose monitoring, not to mention gas analysis. Moreover, such technology requires customized epitaxial growth and electron-beam lithography based post-processing, which inevitably results in low yields and high cost at current technology readiness level. Similar concerns also apply to those designs based on colloidal quantum dot18 and perovskite quantum dot19 given the complexities of their fabrication processes.In principle, the miniaturization of spectrometers inevitably leads to performance trade-offs in size, operating bandwidth, measuring speed, spectral resolution, dynamic range (or insertion loss), etc. Thus, to achieve commercial products, it is vital to tailor the spectrometer design to suit specific application scenarios. By now, a wide variety of miniaturized spectrometers have been demonstrated, involving diverse architectures, working principles and material platforms, as well as varying technology readiness levels for large-scale manufacturing21. Besides, the silicon photonics platform features an ultra-high refractive index contrast, facilitating the realization of small device footprints. These advantages can be further extended by introducing other CMOS compatible materials, such as, silicon nitride for its ultra-low propagation loss, broadband transparent spectral window, and insensitivity to temperature variations22. Furthermore, by means of heterogeneous integration approaches, III-V gain materials can be heterogeneously or monolithically integrated within the same chip package to implement high-performance light sources and photodetectors24, enabling densely integrated spectrometer on-chip systems.To ensure low cost, miniaturized spectrometers must demonstrate a path toward high-volume manufacturing. This generally means lithography-based fabrication and high-level integration. Being compatible with the mature CMOS technologies, silicon photonics provides a promising solutionDifferent to other review articles summarizing the general trends on miniaturized spectrometers, this paper pays special attention to the integrated spectrometers that hold great promises for massive fabrication at low cost and specifies their application perspectives. In the following chapter, we will first give a brief market analysis of miniaturized spectrometers and in the chapter after that, application-oriented analysis of miniaturized spectrometers will be provided, including bio-medical monitoring and industrial sensing. In chapter 4, an in-depth technological analysis of miniaturized spectrometers that can be potentially massively manufactured will be performed. The outlook toward developing next-generation integrated spectrometer will be given in chapter 5. Finally, we draw the conclusions in chapter 6.26. The predicted penetration rates and market values of compact spectrometers in different applications and segments are shown in Fig. 19. The research field has the largest value in 2019 whereas the rapid growth occurs at consumer and biomedical markets almost from 300 to 400% within five years.Due to the unique light-matter interaction, optical spectroscopy has proven to be an efficient technique to non-invasively analyze compositions of chemical materials, gases, biological tissues, food, etc. The applications of spectrometers were predominantly limited to labs, tests, and metrology, whilst they are now sharply penetrating to consumer market in healthcare monitoring, and food safety. This trend is largely attributed to the development of miniaturized spectrometers, as evident by the rapid growth of the market volume of micro- and nano-spectrometers which arises from 655 million US dollars to 922 million in just three years between 2016 and 201927. The rapidly expanding market imposes strong stimulations for both academia and industry players to join this field. Hamamatsu launched a mini spectrometer with a size comparable to fingertip and a weight of only 0.3 g. The device works between 650 nm and 1050 nm with a spectral resolution of about 15 nm, which is suitable for food inspection, light-level measurement and component analysis28. NeoSpectra-Micro developed an integrated near infrared spectral sensor (1250–2500 nm) with 16 nm resolution that can be used in a wide variety of material sensing applications for both qualitative and quantitative analysis29. Continuous efforts are also being poured into developing low-cost chip-scale spectrometers that can be integrated into consumer electronics such as smartphones. For instance, Fraunhofer-ENAS announced its goal of batch producing “one gram spectrometer” for smartphone integration at a cost of around one dollar10. Samsung patented their smartphone-embedded IR spectrometers and prepared to integrate them in Galaxy S11 for skin moisture detection and CO2 measurement27. In 2020, trinamiX and Viavi Solutions had announced a joint force in building a near infrared spectrometer module targeting for consumer devices. Very recently, Rockley Photonics, a silicon photonics enterprise focusing on telecommunications, released a whitepaper about highly manufacturable on-chip spectrometers for “clinic on the wrist” health monitoring11.Among those micro- and nano-spectrometers, chip-scale spectrometers are shown to play a dominant role in the rapid market growth in coming years, due to their superior integrability into portable devices such as smartphones, wearable devices and drones, which, on the other hand, is the main driving force behind the development of compact spectrometers. A recent report foresees that chip-scale spectrometers will have a disruptive increase in market volume from less than 2 million US dollars in 2019 to over 1.6 billion US dollars in 2024, as shown in Fig. 2All these examples herald the rapid development of chip-scale spectrometers in the future, especially for the consumer and biomedical applications. It is foreseeable that the next-generation integrated spectrometers will be developed on the basis of silicon photonics to enjoy massive productibility at low cost and targeted for the adaptability in ecosystems of intelligent devices such as smartphone and wearable devices4, while for the detection of biomarkers, such as blood glucose or lactate, a sub-nanometer resolution is desired to identify their wide signature absorption peaks (~30 nm) to ensure the measurement accuracy31. Therefore, it is critical to establish a set of key figures and specify their application-oriented metrics. In this chapter, we discuss the application prospects for integrated spectrometers in different fields, elaborating their key figures of merits in terms of resolution, bandwidth, etc.Chip-size integrated spectrometers offer great potential for large-scale, in-field, and real-time spectroscopic sensing. Nevertheless, in general, the footprint reduction of a spectrometer consequentially give rise to certain performance degradation regarding its operation bandwidth, resolution, measuring speed, dynamic range, or signal-to-noise ratio, making it crucial to customize the spectrometer design for specified application scenarios. For example, in some cases such as the portable spectroscopies for chemical sensing, a resolution on the scale of 10 nm can be considered acceptable31, i.e., to recognize certain chemical substances or functional groups based on the interaction between electromagnetic radiation with vibrational molecules. Among them, since the MIR technique is not suitable for on-chip integration due to the lack of appropriate integration platforms , it is not within the scope of this paper. Here we focus on the NIR and Raman spectroscopy techniques that are applicable for chip-size integrated spectrometers and summarize their key spectroscopic performance metrics required for different application scenarios.By now, the most popular but demanding application scenarios of the integrated optical spectrometers include biomedical sensing for health monitoring and disease diagnosing, chemical sensing for pollution monitoring, gas leak detection and industrial emission screening etc. These detections rely on different vibrational spectroscopies such as near-infrared , mid-infrared , and Raman spectroscopy  techniques to measure the relevant markers35. For instance, the detection of the glucose concentration in blood or saliva can provide diagnostic or monitoring information for diabetes, while measuring the electrolyte level in sweat helps screen for dehydration. These biomedical measurements are commonly achieved by NIR spectroscopy techniques. Since the absorption bands in NIR region are prominently generated by a large number of overtones and combinations of fundamental vibrations of -CH, -NH, and -OH functional groups, the information of various biomarkers can therefore be elucidated accordingly. Benefitting from the maturity of spectrometer integration platform, NIR spectroscopy with portable devices has gradually become a research hotspot, showing promising prospects for in-vivo biomedical sensing.The sensing of biomarkers in either exhaled breath or biofluids, e.g., blood, breath, sweat, saliva, urine, is of great importance for bioanalysis and biomedical diagnosis36. In theory, by identifying the spectral signatures at different bands, quantitative information of corresponded chemical compounds can be retrieved, e.g., the overtones of -CH stretch are associated with carbohydrates while the overtones of -NH stretch relate to proteins. However, given that these peaks in NIR region are rather broad and do overlap, specified spectrometer metrics, as well as additional calibration processes, such as partial least squares regression (PLSR), are needed to extract the mixed bio-information with reasonable accuracy38. Specifically, such NIR spectrometers should be designed to feature high resolution in particular wavelength regions where the minimum peak overlaps with water or other molecules exist, as well as high sensitivity since the measurable light (either reflected by or transmitted through living tissues) is always rather weak.Figure 45. The informative peaks of the glucose molecule come from the vibrations between -OH and -CH in either long wavelength-NIR  or short wavelength-NIR , representing the two main observe windows on which current analytical methods are based. Relatively, the spectral signatures in LW-NIR are more pronounced than those in SW-NIR which enable higher precision for in-vitro measurement, while the absorption index in SW-NIR bands is notably lower, allowing a deeper penetration depth for in-vivo testing. In Fig. We first take the measurement of blood glucose as a representative example. As one of the earliest applications of NIR spectroscopy in medical diagnosis, researchers have shown great interests in non-invasive systems for in-vivo blood glucose measurements to alleviate the pain and high cost of conventional invasive methods52. Since these compounds have lower concentrations in blood or cells compared to glucose, current measurement approaches are mostly based on in-vitro sample testing, which rely on several hundreds of nanometers wide bandwidths with about 1 nm resolutions in the LW-NIR range. Note that in ref. 53, the detection of deoxygenated hemoglobin (Hb) oxygenated hemoglobin (HbO2) applies only two wavelengths at 730 nm and 850 nm because the absorption spectra at these wavelengths differ significantly between Hb and HbO2, and the ratio is then calculated to determine the concentrations. However, this measurement is not accurate on the patients with abnormal hemoglobin structure, abnormal hemoglobin levels.Besides glucose, NIR spectroscopy has also been demonstrated to extract information about other metabolites, including lactate, urea, glutamine, Ammonia, and (de) oxygenated hemoglobin etc.The above performance requirements can already be met by some commercialized mini spectrometers but are still quite challenging for chip-scale integrated spectrometers. To realize wearable non-invasive devices for in-vivo biomedical sensing, further evolvements in both instrumentation and spectral analysis algorithms are desired.51. For instance, the strong infrared absorption of water (H2O) always severely interferes the spectroscopic detection and limits the scope of applications whilst Raman spectroscopy is insensitive to water. Therefore, for chip-scale spectrometer, NIR and Raman (800~2500 nm) and their combination spectroscopies are well competent for realizing the high-efficiency chemical detection in a great variety of complicated industrial scenarios59.Besides biomedical applications, optical spectroscopic sensing has also been proven as a versatile and powerful tool for high-efficiency industrial chemical detection of various substances. Among them, NIR and Raman spectroscopy are widely adopted and are regarded as a couple of complementary techniques60. So far, the gas detection has been widely demanded by industrial applications, including monitoring the hazardous gases for production safety management, detecting the emission of greenhouse gases and volatile organic compounds (VOCs) for environmental protection61. It can be concluded that for industrial applications except agriculture and food analysis, it is always desirable for the spectrometers to have very high resolution (<1 nm) with a bandwidth over 100 nm, which, as mentioned above, remains a great challenge for integrated spectrometers at this moment.Figure To summarize, the key spectroscopic requirements for both biomedical and industrial chemical sensing applications are listed in Table From the perspective of fundamental technology route, the optical spectrometers can be roughly classified into two groups: wavelength de-multiplexing based (WdM) and wavelength multiplexing (WM) based. As suggested by their names, the WdM spectrometers need to de-multiplex, or in other words, split the incident signals’ spectra, either spatially or temporally, and measure the intensity of individual channel. On-chip WdM spectrometers are typically implemented by dispersive structures, in which different wavelengths exhibit different propagation properties and consequently arrive at detectors distributed at different spatial locations or reach a single detector at different time. Alternatively, WdM spectrometers can be implemented by using an array of narrowband filters or a single tunable narrowband filter, whose spectral responses determine the spectral contents arriving at the detector, as shown in Fig. Being the most straightforward spectroscopic approach, integrated WdM spectrometers don’t require complex computational sources, thus have attracted numerous research attention to be demonstrated on silicon photonics platform. Till now, gratings are the most widely adopted elements for dispersive structures based WdM spectrometers. The fundamental principle is to create multi-path interference effects between the incident signal and make different wavelengths arrive at different locations. Depending on how the multi-path interference is created, gratings based spectrometers can be further divided into planar concave grating (PCG) and arrayed waveguide grating (AWG).62. The main research focus since then have been given to improve the spectrometer’s performance by optimizing the gratings, including using Bragg reflectors64 or photonics crystal reflectors65, coating grating with silver films66, Retro-reflecting V-shaped facets67. etc., as plotted in Fig. PCG is the planar version of the classic diffraction grating spectrometer . The light enters from an input aperture into the free propagation region (a slab waveguide section) after which it diffracts and hits the curved grating on the other end, which reflects as well as focuses different wavelengths at different output apertures located along a so-called Rowland circle as shown in Fig. 40. Compared with PCG, AWG is relatively simpler in design and resolution improvement. However, it has larger footprint as it incorporates two free propagation regions and an array of long waveguides. AWG is also more vulnerable to fabrication variations that induce phase errors along the arrayed waveguides, this is why AWG typically exhibit stronger channel crosstalk than PCG. Various innovations have been created to combat those problems. For instance, the footprint reduction can be achieved by replacing the free propagation regions with multi-mode interferometers  with varying armlength differences, spectrometers with spectral resolutions from 0.13 nm to 20 nm can be easily synthesized81.Another unique advantage of narrowband filter based spectrometer compared with dispersive gratings lies in the tunability of its operation wavelength, particularly for resonant filters, which makes scanning spectrometer that uses a single filter with ultra-compact footprint and large dynamic range possible. However, for filters with very narrow linewidth , tuning the wavelength for a broad optical range will require a significant amount of power consumption and the range will typically be limited by its free spectral range. Therefore, the trend of combining dispersive gratings and narrowband filters for spectrometers with both broad bandwidth, high spectral resolution and small channel count is arising. The dispersive gratings are intended to have low-resolution and split the spectrum into multiple channels with large bandwidth, while a narrowband filter finely scan each entire channel by tuning its center wavelength. Recently, the combination of a AWG and ring resonators was demonstrated for 0.1 nm resolution and 25.4 nm bandwidth with only 9 channels as shown in Fig. The fundamental principle of WdM spectrometers requires the spectrum to be split into multiple channels, which limits the power at each detector and the dynamic range of the spectrometer. In contrast, WM spectrometers pre-process the entire spectrum of the incident signal by certain type of modulation and reconstruct the spectrum using proper algorithms to post-process the modulated spectrum. Based on the specific modulation, WM based spectrometers can be categorized as Fourier transform spectrometers (FTSs) and Computational spectrometers (CSs).82. Compared with WdM spectrometers that commonly measure different spectral components separately in the temporal or spatial domain, the multiplexing measurement of FTSs can provide much better SNR with higher optical throughput captured by the detectors, especially when the detector noise is dominant, which is identified as the Fellgett’s advantage (multiplex) and the Jacquinot’s advantage . According to the Rayleigh criterion, the spectral resolution δλ and bandwidth Δλ of FTSs are determined by the maximum OPD introduced to the signal and the number of OPDs, respectively. For conventional free-spaced FTSs, the OPD modulation can be easily performed using a movable mirror as shown in Fig. FTSs modulate the incident light via tuning the optical path difference (OPD) to obtain the interferograms and then convert them back to the wavelength domain by performing Fourier transform in the post-processing stage, as shown in Fig. 10a83 implements 32 MZIs for a resolution of 0.04 nm, but the bandwidth is limited to only 0.75 nm. The degree of polarization is exploited to further enhance its performance at a given channel count. A Dual-polarization SH-FTS that can process both fundamental transverse electric and transverse magnetic modes was successfully demonstrated in 201915. In detail, the dual-polarized SH-FTS is composed of MZIs with different waveguide widths and arm imbalances, where each MZI can contribute two sampling OPDs, one from the TE mode and the other from the TM mode. As a consequence, the same number of MZIs can provide twice OPD compared with a single polarization operation, as shown in Fig. 84 and machine learning85.SH-FTS are based on a simple and stable multi-interferometer configuration composed of a spatial array of imbalanced MZIs, each of which produce a linearly increasing amount of OPD to the signal, together they could produce a digitized interferogram of the input signal. This kind of configuration can easily produce a very large OPD that turns into high spectral resolution, but it suffers from a similar problem with WdM based spectrometers: inherent trade-off between spectral performance  and channel count. For example, the demonstration in ref. 86, as shown in Fig. 16. After that, the research attention has been given to further increase its resolution. The fundamental reasons for this low resolution include short waveguide length and low power injection to the heater. Increasing waveguide length will result in large propagation loss and high electrical resistance for the heater. By using advanced waveguide design to reduce the optical loss as well as heater structures to receive higher power injection, Li et al. successfully increase the waveguide length from 3 cm in ref. 16 to 10 cm in ref. 87, and the spectral resolution is increased to sub-nanometer. Later, Li et al. combines multiple techniques to further increase the resolution to 0.16 nm, including Michelson interferometers (MI) structure to double the OPD, an optimized heater and air trenches to achieve higher thermal efficiency, a novel multiple interferometers approach is employed which combines balanced MI with N statically imbalanced MIs, thereby increasing the OPD of a single MI by factor of N + 1, as shown in Fig. 10f17.The multi-interferometer configuration with a single-aperture input allows the single-shot interference capture for rapid spectral measurement, while sacrificing the Jacquinot (throughput) advantage. Since the incident light is required to be split equally into every MZI through tree-structured cascaded Y-junctions, the actual throughput for interference modulation at every OPD dramatically decreases with the interferometer number. Temporal heterodyne FTS (TH-FTS), on the other hand, is more analogous to conventional free-space based FTS, as the OPDs are modulated in time domain, and a single detector is used to capture the interferogram. The measurement time is sacrificed in order to maintain Jacquinot (throughput) advantage. Recently, two different mechanisms of achieving temporal modulation of the OPD are demonstrated. First one is similar with SH-FTS in the sense that the OPDs are also in the form of varying physical length difference, but optical switches are employed to dynamically change the physical pathway and realize the temporal modulation of OPD1 and λM refer to the starting and ending wavelength of the spectrum, respectively. For signal processing purpose, the equation can be discretized asRecently, a new scheme of WM based spectrometer named computational spectrometer has drawn extensive study, which utilizes an array of propagation channels with distinct spectral responses, each of which can be interpreted as sampling coefficients of the incident spectrum at each wavelength point, as shown in Fig. Similarly, if the signal passes through N channels with distinct F, the equation has the form ofNxM, this equation can still be reasonably solved at the case N ≪ M, obeying the following rule:For narrowband filters based spectrometer, F is a matrix with only one nonzero component at each row, thus the equation can only be solved when N = M. While for computational spectrometers, with advanced algorithms and proper F88. In 1997 a holographic-sensor was designed using random gratings89. Applying this concept in signal acquisition and imaging processing has also been a well-established technique starting from early of 2000, identified as compressed sensing90. In the field of optical spectrometer, this concept was originally adopted only to combat with the non-ideality of narrowband filters94. In other words, these spectrometers still consist of narrowband filters, but optical span outside filters’ passbands is not zero due to fabrication imperfections, causing unwanted sampling at other wavelength span. Only till 2013, researchers theoretically verified the possibility to employ intentionally designed filters with random transmittances for improving spectrometer’s performance, as shown in Fig. 11b95. The main efforts since then have been put to find integrated filters or dispersive media with desirable spectral responses in order to provide good performance (large M) with only a small number of channels . Generally speaking, the channel spectral responses should meet following requirements:Each channel’s spectral response should be linearly independent with the others to provide uncorrelated spectrum sampling.Each channel’s spectral response should contain adequate sharp features to provide high sampling resolution as the minimum optical distance between two distinguishable points in the spectral response determines the spectral resolution of this filter.The channels should be compact, low loss and be able to enjoy massive production.Using this concept for reconstructing signals in a specific domain is not novel. Back in 1987, researchers already proposed to design a velocimeter (a device reconstructing velocities) using random patterns13. Thanks to its high level of randomness, the first two requirements were met and the spectrometer with about 30 channels can produce a spectral resolution of 0.75 nm within 25 nm bandwidth. The use of silicon photonics indeed enables massive production for such spectrometer, but the large amount of air holes introduces significant loss to the signal  for sub-nm resolution, which is unacceptable for consumer electronics.99, the total detection time for such path-reconfigurable spectrometer can be within one second even for hundreds of detection channels.Computational spectrometers are believed promising for achieving a balanced performance with compact footprint and low power consumption, as it can significantly reduce the number of channels compared with dispersive gratings or narrowband filters based spectrometer, and do not demand power consumption compared with tunable filters spectrometers or TH-FTS. But current demonstrations still require the incident signal to be split over 30~50 sampling channels and the channel count has to scale up in order to achieve better performance, which reduces the power intensity in each channel, resulting in a worse SNR and smaller dynamic range. One potential solution is to replace the passive optical power splitting with active path scanning, that is, to actively configure different optical paths (sampling channels) for the incident signal over time, rather than splitting it over space, as shown in Fig. 101, which is an emerging technology allowing the on-chip optical signal to be manipulated at run-time to enable various applications. As shown in Fig. Currently demonstrated spectrometers, irrespective of platforms and working principles, are all fixed in terms of performance, such as the center wavelength, operation bandwidth and spectral resolution. While it is highly desirable to develop a universal spectrometer that can dynamically tune its performance in order to suit requirements of different applications or working scenarios. Ideally, all of the spectrometers’ performance indicators are desired to be programmable, while in practice, it would be valuable enough to develop a spectrometer with adjustable bandwidth and resolution. This idea can be possibly realized in incorporation with programmable photonic integrated circuits (PICs)103, can also be explored to enhance spectrometer’s performance. Currently, WdM is widely adopted for on-chip optical communication to increase the data volume and has been proved to be able to cover a broad bandwidth, such as C+L-band WdM104 and O+C-band WdM105. Analogically, parallelism can also be introduced into the design of spectrometer systems as shown in Fig. 107.Parallelism, which has always existed in the design of optical transmission system since the arise of wavelength division multiplexing (WdM) technologies109. III-V materials, in contrast, feature direct and tunable bandgap, thus can achieve high absorption coefficients at different spectral bands. Moreover, they can also serve as gain materials for optical emitters, such as lasers110 or LEDs111. Currently, high-density cost-efficient III-V-on-Silicon integration relies on hybrid integration approaches, such as, flip-chip bonding112, die/wafer-to-wafer bonding113, and micro-transfer printing114. Therefore, it is predictable that hybrid integrated platforms will play an important role in the future commercialization of integrated spectrometers.Besides the design of spectrometer system itself, to realize full functionalities, it is desired to also implement high-performance light sources and photon detectors within the same chip package, which requires the introduction of other materials on the basis of silicon photonics. Currently, the mainstream integrated photodetectors are based on germanium, as it’s already a standard fabrication process in many CMOS foundries. However, Ge-on-Si photon detectors always suffer from high dark currents and low absorptivity attributed to its nature of indirect bandgap, and normally cannot cover the spectral range beyond NIR116. An additional design dimension could bring unique advantages for 3D nano-printed spectrometers, such as ultra-compact footprint. A wide operation bandwidth can also be expected thanks to the various ultra-broadband 3D optical coupling strategies117. However, at current stage, 3D nano printing only supports passive polymer structures, making external light sources and photo detectors indispensable. Therefore, by implanting the 3D nano-printed spectrometer onto the hybrid integrated III-V-on-Silicon PICs, it is possible to enjoy the advantages from both sides, representing a promising development direction.Beyond silicon photonic platforms, polymer-based 3D lithography offers new possibilities for massive producible miniaturized spectrometers. Based on the laser-induced polymerization effect, current commercial 3D nano printer can implement arbitrary three-dimensional pattern with <200 nm feature size. A variety of demonstrations of 3D-nanoprinted spectrometers have been reported based on different principles ranging from free-space optics to computational reconsctructionWith the rapid development of Internet-of-Things and popularity of intelligent portable device, the demand for low-cost, lightweight, miniaturized spectrometers has never been so imperative. Various efforts have been made to miniaturize spectrometers, yet they somewhat lack technological readiness for commercial products. Being compatible with CMOS technology, silicon photonics is widely accepted to be one of the most promising platforms for mass-manufacturable photonic integrated circuits. Therefore, in this paper we review recent progresses in integrated spectrometers that leverage silicon photonics technology, including their market trends, application-oriented requirements with a special focus on biomedical sensing and industrial detections, and technological evolution that covers mainstream structures for silicon integrated spectrometers. Four possible research directions are foreseen for developing next-generation integrated spectrometers. We expect to see expanding integration of chip-scale spectrometers into consumer productions within the next few years, providing cost-effective and reliable services to users worldwide.


In this short review, we summarized the results obtained with an assay to detect influenza virus-specific antibodies that mediate ADCC, which was developed and evaluated within the framework of the IMI-funded project “FLUCOP”. HA-specific ADCC mediating antibodies were detected in serum samples from children and adults pre- and post-vaccination with monovalent, trivalent, or quadrivalent seasonal influenza vaccines, or following infection with H1N1pdm09 virus. Additionally, using chimeric influenza HA proteins, the presence of HA-stalk-specific ADCC mediating antibodies after vaccination and natural infection with H1N1pdm09 virus was demonstrated. With serum samples obtained from children that experienced a primary infection with an influenza B virus, we showed that primary infection induces HA-specific ADCC-mediating antibodies that cross-reacted with HA from influenza B viruses from the heterologous lineage. These cross-reactive antibodies were found to be directed to the HA stalk region. Antibodies directed to the influenza B virus HA head mediated low levels of ADCC. Finally, vaccination with a recombinant modified vaccinia virus Ankara expressing the HA gene of a clade 1 A(H5N1) highly pathogenic avian influenza virus led to the induction of ADCC-mediating antibodies, which cross-reacted with H5 viruses of antigenically distinct clades. Taken together, it is clear that virus-specific antibodies induced by infection or vaccination have immunological functionalities in addition to neutralization. These functionalities could contribute to protective immunity. The functional profiling of vaccine-induced antibodies may provide further insight into the effector functions of virus-specific antibodies and their contribution to virus-specific immunity. These outbreaks of influenza are caused by influenza A viruses (IAV) of the H1N1 and H3N2 subtypes, and influenza B virus (IBV). Two lineages of IBV have been distinguished that have cocirculated since the 1970s, the B/Victoria/2/87-like (B/Vic) lineage and the B/Yamagata/16/88-like (B/Yam) lineage, which differ based on genetic and antigenic properties of their hemagglutinins  by seasonal influenza viruses, which is known as antigenic drift. Because HA is the main target for the development of virus-neutralizing (VN) antibodies and the major component of influenza vaccines, the composition of the vaccines needs to be updated almost annually to guarantee optimal effectiveness . The curUpon influenza virus infection, virus-specific antibody and T cell responses are induced. Virus-neutralizing antibodies directed to the viral HA are considered an important correlate of protection from infection, provided that they match the influenza virus causing the infection antigenically. For the detection and quantification of virus-specific serum antibodies the hemagglutination inhibition (HI) and VN assays are commonly used. HI antibody titers are an accepted proxy for VN antibodies and are used to determine the efficacy of seasonal influenza vaccines. HI antibody titers are considered a good correlate of protection , althougvia a different mechanism than preventing receptor interaction, namely by preventing conformational changes of HA that take place in the endosome in a pH dependent fashion. These antibodies additionally exert other biological activities that contribute to protective immunity, which are described below. Because the HA-stalk is relatively conserved, even across various subtypes of IAV, stalk-specific antibodies are highly cross-reactive.VN antibodies are typically directed to epitopes located in and around the RBD of the variable globular head domain of HA and thus can block binding of the virus to its receptor on host cells. Most HI antibodies are strain-specific and do not recognize variants that have accumulated one or more amino acid substitution in vicinity of the RBD . Becausevia Fc-receptors and contribute to protective antiviral immunity, as was shown for influenza virus , antibody-dependent complement deposition (ADC) and antibody dependent cellular cytotoxicity (ADCC). All of these activities are mediated za virus –9, humanza virus –12, and za virus –15. The In the framework of the Innovative Medicines Initiative (IMI) funded project “FLUCOP”, which aimed at the standardization and development of assays for assessment of influenza vaccines correlates of protection, we developed and used an assay for the detection of antibodies with ADCC activity that would be suitable for standardization. Here, we compiled antibody-dependent cellular cytotoxicity (ADCC) data obtained in four separate studies to get a comprehensive overview of the induction of ADCC-mediating antibodies after influenza virus infection, or immunization with monovalent, trivalent, or quadrivalent influenza vaccines –19.22.1All data shown here were obtained with serum samples from subjects that were previously enrolled in other studies; ethical permission was therefore previously obtained.Serum samples were obtained from 9- and 10-year-old children before and after infection with the H1N1pdm09 virus  . In addiThe serum panels included in this study are summarized in 2.2Mycoplasma species, and were only cultured for a limited number of passages. In brief, microtiter plates were coated with recombinant protein  and after blocking the plates with bovine serum albumin (BSA), specific serum antibodies were allowed to bind at a serum dilution of 1:160. This dilution was found to be optimal and in the linear range of the dose response curve. A serum pool from uninfected children, intravenous immunoglobulins  and a high-positive serum were included in the experiments as negative and positive controls. After the serum incubation, 1x10(5) NK92.05-CD16 cells were added per well in the presence of a monoclonal antibody to CD107a, (degranulation marker), golgistop and golgiplug. After incubation for 5 hours at 37°C, the cells were stained for flow cytometric analysis. Live NK92.05-CD16 cells were identified by LIVE/DEAD and CD56 staining as described previously; CD56-PE (BD Biosciences) was used in a concentration of 1µg/ml, aqua live/dead (Thermo Fisher) was used per manufacturer’s instruction  as previously described . NK92.05truction , 18. Sub2.3For the detection of ADCC-mediating antibodies to influenza A and B viruses, several antigens were used as previously described –19. Puri2.4Differences between two groups were assessed by either Mann-Whitney U test or Wilcoxon rank test, dependent on whether unpaired or paired samples were used. Differences between multiple groups were assessed by Kruskal-Wallis test with multiple comparisons. Linear regression on log-transformed ADNK activation percentages was performed for the serum samples from MVA-H5 vaccinated participants.33.1To get a complete overview of the induction of ADCC-mediating antibodies by influenza virus infection or influenza vaccination, we compiled datasets from four different studies Table 1.3.2To determine whether a primary infection with influenza virus induced ADCC-mediating antibodies, ADCC was measured in a unique sample set of sera from 5 children obtained before and after primary infection with H1N1pdm09 influenza virus , and selected study subjects were boosted with an additional vaccine 1 year later. Serum samples were obtained prior to vaccination and throughout the trial. ADCC-mediating antibodies targeting the full-length homologous H5 protein (A/Vietnam/1194/04) were detected prior to vaccination in some participants, although exposure to an H5 IAV was not deemed likely . By measuring ADCC-mediating antibodies after adjuvanted inactivated trivalent vaccination in 24 adults  in combination with recombinant proteins bound to a solid phase. Activation of NK cells cination . The usecination , 30.We developed this assay in the framework of the IMI project “FLUCOP”, aimed at the standardization and development of assays for assessment of influenza vaccines correlates of protection. Although we found that results obtained with the developed ADCC degranulation assay were highly reproducible, the assay still depended on biological components (NK cells) and is relatively low-throughput. For that reason, reporter assays based on cells that express luciferase upon engagement of FcγRIIIa have been developed. Recently, results obtained with the degranulation assay were compared with those obtained with a reporter assay, which showed that the results strongly and significantly correlated . The repIt is clear that virus-specific antibodies, induced by either infection or vaccination, have immunologically relevant functionalities, in addition to neutralization of virus infectivity by blocking glycoprotein – receptor interactions; ADP-, ADC- and ADCC-mediating antibodies are likely to contribute to protective immunity , 10–15. The importance of functionality profiling was recently demonstrated in a phase I clinical trial, in which chimeric hemagglutinin-based universal influenza virus vaccines were used to induce broad immunity. This was achieved by targeted boosting of HA-stalk-specific antibodies; because the HA stalk is conserved, these antibodies contribute to heterosubtypic immunity. In this trial, not only did the authors demonstrate the presence of broadly-reactive stalk-specific antibodies, they also showed functionality in neuraminidase inhibition and ADCC reporter assays. Passive transfer of these antibodies in mice showed that they were indeed an independent correlate of protection . The indTogether, we showed that influenza virus infection and influenza vaccination led to the induction of ADCC-mediating antibodies, which can be rapidly recalled by re-infection or re-vaccination. ADCC-mediating antibodies often target epitopes in the relatively conserved HA-stalk, potentially important to achieve broadly-reactive immunity. We conclude that complete functional profiling of the antibody responses is crucial to provide insight into effector functions of virus-specific antibodies and their contribution to virus-specific immunity. In addition to profiling antibody responses in blood, it is important to additionally assess the contribution of IgA at the mucosal sites. The availability of reliable assays for the measurement of functional antibodies will aid the evaluation of existing and novel influenza vaccines.RDdV and GFR conceptualized the manuscript. KH organised clinical samples for the study. All authors contributed to the writing of the manuscript. KH contributed to the study design.


Life-space mobility (LSM), as the extent of mobility within one’s environment, is a key for successful aging and has become a relevant concept in gerontology and geriatric research. Adequate assessment instruments are needed to identify older persons with LSM restrictions, and to initiate, adapt or evaluate intervention strategies.To systematically identify, describe and analyze the psychometric properties of LSM questionnaires, with a special focus on their availability in the German language.A systematic literature search was conducted in PubMed, PsycINFO, Cochrane Library, CINAHL, and Web of Science. Studies that examined at least one psychometric property of LSM questionnaires published up to August 2021 were included and evaluated based on the consensus-based standards for the selection of health measurement instruments (COSMIN) guidelines.This study included 37 validation studies describing 13 different LSM questionnaires. Methodological quality and comprehensiveness of validations were heterogeneous. Based on comprehensive and high-quality results, four LSM questionnaires stood out: the University of Alabama at Birmingham life-space assessment (UAB-LSA), life-space assessment in persons with cognitive impairment (LSA-CI), interview-based and proxy-based versions of the life-space assessment in institutionalized settings (LSA-IS), all of them available in the German language.This systematic review provides a concise overview of available LSM questionnaires and their psychometric properties to facilitate the selection for use in clinical practice and research. The UAB-LSA and LSA-CI for community settings and the interview-based or proxy-based LSA-IS for institutional settings were found to be the most appropriate LSM questionnaires.The online version of this article (10.1007/s00391-022-02035-5) contains supplementary material, which is available to authorized users. Mobility is a key factor for successful aging. In clinical practice and research, assessing mobility is important for identifying individuals at risk or with mobility limitations, developing and adapting intervention strategies, and evaluating intervention effectiveness. Various mobility instruments have been developed, with a focus on assessing motor capacity ; howeverDespite technical developments such as the global positioning system (GPS) to objectively measure life-space parameters , LSM hasSelecting an appropriate questionnaire for clinical practice and research is challenging. Setting and target population as well as practical aspects , and the methodological quality of the instrument determined by psychometric properties  must be considered. The use of instruments validated in the appropriate language and cultural setting represents a mandatory methodological criterion, which has so far not been documented for LSM questionnaires in the German language. In addition, previous reviews of LSM measurement tools focused only on specific LSM questionnaires  or did nThus, the primary aim of the present systematic review was to provide an overview on currently available LSM questionnaires and their psychometric properties to facilitate the selection for use in clinical practice and research. A secondary aim was to identify LSM questionnaires validated for use in the German language.A systematic literature search was conducted in accordance with recommendations from the Preferred Reporting Items for Systematic review and Meta-Analysis (PRISMA) protocols  and the We performed a complete database search with no language restrictions in CINAHL, Cochrane Library, PsycInfo, Pubmed, and Web of Science up to August 2021. Search terms included combinations of variants for the keyword life-space along with different terms for assessment, validation, and psychometric properties (see Table S1), as recommended elsewhere .Titles, abstracts and full texts of studies identified by the search were screened for eligibility by two independent reviewers . Any disagreements were solved by a third reviewer (KH). Reference lists of relevant articles and reviews, and grey literature were also screened for additional studies for inclusion.Studies eligible for inclusion provided empirical evidence for validity, reliability, sensitivity to change, and/or feasibility of a questionnaire designed to assess more than two spatial aspects of mobility (e.g. not only indoor vs. outdoor), usually described as life-space mobility (LSM). Each version of a LSM questionnaire with relevant changes to the assessment procedure or the information collected were considered as a separate instrument.Data extraction was performed by two independent reviewers . Information on main features of the identified questionnaires  was extracted from the original article of each questionnaire. Information on different aspects of feasibility, validity, reliability, and sensitivity to change was gathered from all included studies. The methodological quality of the included studies was rated by three reviewers  using the COSMIN risk of bias checklist, considering additional references for sample size rating , 11. DisAn additional focus of the narrative analysis was lain on the availability of LSM questionnaires validated in the German language.The literature search yielded 37 studies eligible for inclusion see Fig. , in whicA detailed description of these questionnaires is provided in Table S2. The majority of them consider not only spatial  but also contextual aspects  of mobility. Nine questionnaires have initially been developed for community-dwelling older persons  –21, 24, n = 19) were identified for evaluating the psychometric properties of UAB-LSA [Figure  UAB-LSA  in diffe UAB-LSA –45, addi UAB-LSA , 46, 47  UAB-LSA  are alsoSample sizes of the validation studies ranged from 5  to 2147 Floor or ceiling effects were analyzed in 16 studies, with all reporting absence of such effects –48, 50. r > 0.5 for similar  and r > 0.30–0.50 for related constructs  for evaluation. Concurrent validity was assessed in seven studies [Almost all studies included a validity analysis. Construct validity was assessed in 30 studies –48, 50,  studies , 49, 50. studies , 24, con studies , 28, 36, studies , 28.Reliability results were evaluated in terms of test-retest reliability in 22 studies, showing predominantly acceptable to good results with intraclass correlation coefficients or Kappas of > 0.7 . Poor teOnly eight studies analyzed sensitivity to change of the LSM questionnaires , 47, 48.The methodological quality of the included validation studies assessing the different psychometric properties was predominantly moderate to high across studies (Table S3). Overall, methodological quality of validity studies was predominantly negatively affected by low sample sizes that were not statistically justified , 42, 50 The LSA-CI and both versions of the LSA-IS were comprehensively validated in the German language, with overall good results on feasibility, validity, reliability, and sensitivity to change –23, 48. This systematic review provides a concise overview of currently available questionnaires to assess LSM and their psychometric properties with comprehensive information in the supplements. To the best of our knowledge, this is the first review to systematically analyze such a wide range of LSM questionnaires, with the aim of providing clinicians and researchers with practical knowledge on LSM questionnaires to help them select an instrument appropriate for their purpose.A large number of different LSM questionnaires were identified. Given the importance of LSM-associated factors with aspects of successful aging  , 15 and Apart from appropriateness of the instrument for the target population, which also covers the availability in the specific language, its feasibility plays an important role in both clinical routine and research. Although feasibility aspects of the LSM questionnaires were rarely reported or only as side notes, presented results for the completion rates/times and floor/ceiling effects suggest that they are accepted and not time consuming and cover an adequate range of respondents’ LSM.Most information was provided for the validity of the LSM questionnaires, more specifically for their construct validity showing predominantly hypothesis-confirming associations with physical, cognitive, psychosocial, financial, environmental, and/or sociodemographic variables. Given the overall complexity of real-life mobility , it was Among the different LSM questionnaires, the UAB-LSA stands out due to its widespread use and the availability of validated versions in various languages and populations. Potential limitations of the UAB-LSA are its relatively long, retrospective assessment period of 4 weeks and its focus on the community setting, which may complicate its use in studies focusing on more short-term LSM (changes), in populations with cognitive impairment due to possible recall bias or institutional settings. To promote recall of LSM, the recommended LSA-CI covers a shorter assessment period of 1 week and has shown to be feasible, valid, reliable and sensitive to intervention-induced changes in community-dwelling older persons with and without cognitive impairment , 48. TheOverall, recommendations for use based on comprehensive results on adequate psychometric properties with high methodological quality could only be identified for the UAB-LSA  and the Future studies on psychometric properties of LSM questionnaires should focus on feasibility aspects, select variables for construct validity analyses based on a conceptual LSM framework and evaluate the ability to detect changes in LSM. Future developments might combine the accuracy of sensor-based assessments with aspects evaluated by questionnaires concerning independence of mobility.Several life-space mobility (LSM) questionnaires are available for clinicians or researchers to assess spatial aspects of mobility in everyday life; however, only four questionnaires provided a comprehensive validation including good feasibility, validity, reliability and sensitivity to change. Given the comprehensive validation and overall good psychometric properties, the UAB-LSA and LSA-CI can be recommended for use in community-dwelling older persons and the two versions of the LSA-IS (interview-based and proxy-based) for use in institutionalized older persons. Each of these four LSM questionnaires is available in the German language.Most identified LSM questionnaires showed acceptable to good psychometric properties, although validation strategies were partly limited to single psychometric properties in a number of studies.Based on the available evidence, we recommend the most comprehensively validated instruments with overall good psychometric properties: UAB-LSA and LSA-CI for community-dwelling older persons and the LSA-IS as self-based or proxy-based version for institutionalized older persons.All recommended questionnaires are validated for use in the German language.The supplemental documents include information on the search terms (Table S1), characteristics (Table S2) and psychometric properties (Table S3) of included assessment instruments, and the available assessment forms and manuals in German language