Rapid tissue differentiation at the molecular level is a prerequisite for precise surgical resection, which is of special value for the treatment of malignant tumors, such as glioblastoma (GBM). Herein, a SERS-active microneedle is prepared by modifying glutathione (GSH)-responsive molecules, 5,5\'-dithiobis(2-nitrobenzoic acid) (DTNB), on the surface of Au@Ag substrates for the distinction of different GBM tissues. Since the Raman signals on the surface of the DTNB@Au@Ag microneedle can be collected by both portable and benchtop Raman spectrometers, the distribution of GSH in different tissues at centimeter scale can be displayed through Raman spectroscopy and Raman imaging, and the entire analysis process can be accomplished within 12 min. Accordingly, in vivo brain tissues of orthotopic GBM xenograft mice and ex vivo tissues of GBM patients are accurately differentiated with the microneedle, and the results are well consistent with tissue staining and postoperative pathological reports. In addition, the outline of tumor, peritumoral, and normal tissues can be indicated by the DTNB@Au@Ag microneedle for at least 56 days. Considering that the tumor tissues are quickly discriminated at the molecular level without the restriction of depth, the DTNB@Au@Ag microneedle is promising to be a powerful intraoperative diagnostic tool for surgery navigation.

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5\'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

Studies have found that matrix metalloproteinase-9 (MMP-9) plays a significant role in cancer cell invasion, metastasis, and tumor growth. But it is a challenge to go for highly sensitive and selective detection and targeting of MMP-9 due to the similar structure and function of the MMP proteins family. Herein, a novel surface-enhanced Raman scattering (SERS) sensing strategy was developed based on the aptamer-induced SERS \"hot spot\" formation for the extremely sensitive and selective determination of MMP-9. To develop the nanosensor, one group of gold nanospheres was modified with MMP-9 aptamer and its complementary strand DNA1, while DNA2 (complementary to DNA1) and the probe molecule 5,5\'-dithiobis-(2-nitrobenzoic acid) (DTNB) were grafted on the surface of the other group of gold nanospheres. In the absence of MMP-9, DTNB located on the 13-nm gold nanospheres has only generated a very weak SERS signal. However, when MMP-9 is present, the aptamer preferentially binds to the MMP-9 to construct MMP-9-aptamer complex. The bare DNA1 can recognize and bind to DNA2, which causes them to move in close proximity and create a SERS hot spot effect. Due to this action, the SERS signal of DTNB located at the nanoparticle gap is greatly enhanced, achieving highly sensitive detection of MMP-9. Since the hot spot effect is caused by the aptamer that specifically recognizes MMP-9, the approach exhibits excellent selectivity for MMP-9 detection. Based on the benefits of both high sensitivity and excellent selectivity, this method was used to distinguish the difference in MMP-9 levels between normal and cancer cells as well as the expression of MMP-9 from cancer cells with different degrees of metastasis. In addition, this strategy can accurately reflect the dynamic changes in intracellular MMP-9 levels, stimulated by the MMP-9 activator and inhibitor. This strategy is expected to be transformed into a new technique for diagnosis of specific cancers related to MMP-9 and assessing the extent of cancer occurrence, development and metastasis.

A lateral flow assay (LFA) strip based on dual 5,5\'-dithiobis-(2-nitrobenzoic acid) (DTNB)-encoded satellite Fe3O4@Au (Mag@Au) SERS tags with nanogap is reported for  ultrasensitive and simultaneous diagnosis of two SARS-CoV-2 functional proteins. Composed of Fe3O4 core, satellite gold shell with nanogaps, and double-layer DTNB, the Mag@Au nanoparticles with an average size of 238 nm were designed as multifunctional tags to efficiently enrich the target SARS-CoV-2 protein from complex samples, significantly enhancing the SERS signal of the LFA strip and provide quantitative SERS detection of analyte on test lines. The developed dual DTNB-encoded satellite Mag@Au-based LFA allowed simultaneous quantification of spike (S) protein and nucleocapsid (NP) protein with detection limits of 23 pg mL-1 and 2 pg mL-1, respectively, lower than commercial ELISA kits and reported SERS-LFA detection system-based Au NPs and Fe3O4@3 nm Au MNPs. This magnetic SERS-LFA also showed high performance of multi-variant strain detection and further distinguished clinical samples of Omicron variant infection, demonstrating the potential of in situ detection of respiratory virus diseases.

BACKGROUND: Histamine is a kind of biogenic amine with strong toxicity and potential carcinogenicity. Many traditional methods of detecting histamine have the disadvantages of cumbersome detection steps, expensive equipment, and high professional requirements for staff. In contrast, SERS has become the preferred method for quantitative analysis of histamine because of rich fingerprint information, rapidity and economy. However, most of SERS substrates still have technical problems, such as poor stability, low sample collection rate, and detection efficiency. Therefore, there is a great need for new strategies to develop high-performance SERS substrates based sensors.
RESULTS: In our study, a sensitive SERS aptasensor for the detection of histamine was synthesized. The assembly was formed between IRMOF-3@Au/PDMS (flexible SERS substrate) and AuNR-DTNB@Ag-HA apt (Raman signal probe with both the target capture ability) via π-π stacking interaction from HA aptamer and IRMOF-3. Consequently, the SERS signal of the assembly derived from DTNB reached highest due to the synergistic enhancement effect by AuNR@Ag and IRMOF-3@Au. Meanwhile, HA aptamer can specifically capture histamine, therefore histamine addition competitively bound to the probe, leading to a corresponding decrease in the DTNB signal value on the SERS substrate. The SERS intensity at 1331 cm-1 presented a good linear relationship towards the logarithmic value of histamine concentrations ranging from 0.0001 mg/L to 400 mg/L (R2 = 0.990) with the LOD of 3.6 × 10-5 mg/L. Furthermore, the application in wine samples demonstrated the accuracy and applicability of the developed sensor.
CONCLUSIONS: This method effectively improves substrate stability, detection sensitivity and signal response immediacy to amplify the SERS sensor, thus satisfying the histamine detection requirements of various systems. According to this aptasensor design, our strategy can be extended to create other MOF-based SERS substrates for accurately detecting relative targets to ensure food safety.

Matrix metalloproteinase 2 (MMP-2) has been considered a promising molecular biomarker for cancer diagnosis due to its related dysregulation. In this work, a core-satellite structure-powered ratiometric surface-enhanced Raman scattering (SERS) nanosensor with high sensitivity and specificity to MMP-2 was developed. The SERS nanosensor was composed of a magnetic bead encapsulated within a 5,5\'-dithiobis(2-nitrobenzoic acid) (DTNB)-labeled gold shell as the capture core and a 4-mercaptobenzonitrile (MBN)-encoded silver nanoparticle as the signal satellite, which were connected through a peptide substrate of MMP-2. MMP-2-triggered cleavage of peptides from the core surface resulted in a decrease of the SERS intensity of MBN. Since the SERS intensity of DTNB was used as an internal standard, the reliable and sensitive quantification of MMP-2 activity would be realized by the ratiometric SERS signal, with a limit of detection as low as 2.067 ng/mL and a dynamic range from 5 to 100 ng/mL. Importantly, the nanosensor enabled a precise determination of MMP-2 activity in tumor cell secretions, which may provide an avenue for early diagnosis and classification of malignant tumors.

Sulfamethazine (SM2) is an antibacterial drug，which has been extensively used in human and veterinary medicine, long-term consumption of which may lead to the accumulation of sulfonamides in the body. Detection of sulfonamides often uses microbiological approaches, mass spectrometry and chromatography, which are expensive and time-consuming. Surface-enhanced Raman scattering-based immunochromatographic assay (SERS-ICA) has been recently applied in the detection. Herein, a Janus-labeled Au nanoparticle with subnanosized SiO2-monoclonal antibody and SERS reporter (DTNB) modified simultaneously (mAbAuNpDTNB) has been developed in a SERS-based lateral flow immunosensor, which can be used for rapid, quantitative and ultrasensitive detection of sulfamethazine residue in milk. The mAbAuNpDTNB exhibits a specific array on a paper stripe, which not only identifies sulfamethazine but also straightforwardly exposes the Raman reporter between the AuNps via self-assembly. The detection sensitivity of SERS-ICA for sulfamethazine reached 0.1 pg/mL, which was far below the previously published value by ELISA and the maximum residue limit set by the European Union. The entire SERS-ICA detection for sulfamethazine was completed within 15 min. Furthermore, high accuracy for this assay was exhibited in the spiking experiment with a recovery percentage of 88.1%-112.7%. The results demonstrated that this SERS-ICA can potentially be applied in point-of-care testing as an ultrasensitive and quantitative to semi-quantitative analytical method.

Au coated magnetic polyphosphazene (MPCTP) composite particles (MPCTP@Au) were fabricated with sensitive SERS activity. The MPCTP particles were generated by coating polyphosphazene on Fe3O4 nanoparticles through precipitation polycondensation of hexachlorocyclotriphosphazene and phloroglucinol. MPCTP@Au composite particles were obtained by deposition of Au nanoparticles on MPCTP by the reduction of HAuCl4. The size and the thickness of the Au shell can be controlled by varying the amount of HAuCl4. The magnetic core endowed the composite particles with good magnetic responsiveness, which allowed the analyte to be enriched and separated from the complex matrix, and significantly simplifying the sample pretreatment procedure. The SERS activity of MPCTP@Au composite particles were evaluated by DTNB as model Raman reporter, and the limits of detection (LOD) of DTNB was 10-8 mol/L. A high efficient SERS immunoassay system based on the MPCTP@Au substrates for the detection of immunoproteins was developed. Human IgG and rabbit IgG were quantitatively determinated simultaneously by this immunoassay system. The quantitative determination of the immunoglobulin G (IgG) was achieved and the LOD of human IgG, rabbit IgG and the mixture of human IgG and rabbit IgG were as low as 10 fg/mL, 100 pg/mL and 1 ng/mL, respectively. The results showed that the MPCTP@Au composite particles have broad application prospects as high performance SERS active substrates for immunoprotein analysis.

BAHD acyl-coenzyme A (CoA) acyltransferases play key roles in a large number of biosynthetic reactions involved in plant specialized metabolism. One approach to measure reaction rates for these enzymes is to quantify the amide or ester reaction products following chromatographic separation of reaction components, an approach that can be labor intensive and time consuming, and complicated by a lack of pure standards. We previously developed and validated an alternative approach using 5,5\'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman\'s reagent) to spectrophotometrically monitor reaction progress by the release of free CoA in the reaction. This approach allows near-real time measurement of reaction rates, permitting reaction conditions (buffer, reactant, and enzyme concentrations, etc.) to be changed \"on the fly.\" The ease and rapidity of data collection allows a high density of data points to be collected for determination of kinetic parameters. Here we provide a detailed procedure for using DTNB to measure BAHD acyl-CoA acyltransferase reaction rates, and as an example, use it to determine kinetic parameters for red clover hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase, a BAHD acyl-CoA hydroxycinnamoyltransferase not previously characterized with respect to kinetic parameters. This approach may be more generally applicable to transferases using CoA donors.

Pheochromocytoma (PCC), a rare tumor, often develops distant metastases after diagnosis, delaying early intervention treatment. In order to overcome the limitations of traditional diagnostic methods, dual-targeting Surface-Enhancement Raman Scattering (SERS) cytosensor was developed to identify and detect PCC-CTCs from peripheral blood. Meta-iodobenzylguanidine (MIBG) and octreotide-2,2\',2″,2\'\'\'- (1,4,7,10 -tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid (DOTA) functionalized magnetic Fe3O4 and Ag-DTNB were prepared as capture probe and signal probe for SERS signal export, respectively. Ag nanocubes (AgNCs) as Raman active substrate offer an enhanced electromagnetic field, which could effectively enhance the signal intensity of DTNB and potentially realize trace analyte detection. The obtained SERS fingerprint spectroscopy possessed the characteristic of high sensitivity and resolution in the concentration range from 3.0-3.0 × 106 cells mL-1, with a detection limit of 1 cell mL-1, which laterally compensated the deficiency of scarce CTCs in peripheral blood. This work provided new insight into PCC-CTCs accurate detection.