ABSTRACT
Spatial relations between tumor cells and host-infiltrating cells are increasingly important in both basic science and clinical research. In this study, we have tested the feasibility of using standard methods of immunohistochemistry (IHC) in a multiplex staining system using a newly developed set of chromogenic substrates for the peroxidase and alkaline phosphatase enzymes. Using this approach, we have developed a set of chromogens characterized by (1) providing fine cellular detail, (2) non-overlapping spectral profiles, (3) an absence of interactions between chromogens, (4) stability when stored, and (5) compatibility with current standard immunohistochemistry practices. When viewed microscopically under brightfield illumination, the chromogens yielded the following colors: red, black, blue, yellow, brown, and green. By selecting compatible color combinations, we have shown feasibility for four-color multiplex staining. Depending on the particular type of analysis being performed, visual analysis, without the aid of computer-assisted image analysis, was sufficient to differentiate up to four different markers.
Subject(s)
Immunohistochemistry , Immunohistochemistry/methods , Humans , Chromogenic Compounds/metabolism , Chromogenic Compounds/chemistry , Staining and Labeling/methodsABSTRACT
RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.
Subject(s)
In Situ Hybridization , RNA , In Situ Hybridization/methods , RNA/genetics , RNA/analysis , Humans , Chromogenic Compounds/chemistry , Colorimetry/methods , Single Molecule Imaging/methodsABSTRACT
Organophosphorus nerve agents (OPNAs) pose a great threat to humanity. Possessing extreme toxicity, rapid lethality, and an unassuming appearance, these chemical warfare agents must be quickly and selectively identified so that treatment can be administered to those affected. Chromogenic detection is the most convenient form of OPNA detection, but current methods suffer from false positives. Here, nitrogenous base adducts of dirhodium(II,II) acetate were synthesized and used as chromogenic detectors of diethyl chlorophosphate (DCP), an OPNA simulant. UV-vis spectrophotometry was used to evaluate the sensitivity and selectivity of the complexes in the detection of DCP. Visual limits of detection (LOD) for DCP were as low as 1.5 mM DCP, while UV-vis-based LODs were as low as 0.113 µM. The dirhodium(II,II) complexes were also tested with several potential interferents, none of which produced a visual color change that could be mistaken for OPNA response. Ultimately, the Rh2(OAc)4(1,8-diazabicyclo[5.4.0]undec-7-ene)2 complex showed the best combination of detection capability and interferent resistance. These results, when taken together, show that dirhodium(II,II) paddlewheel complexes with nitrogenous base adducts can produce instant, selective, and sensitive detection of DCP. It is our aim to further explore and apply this new motif to produce even more capable OPNA sensors.
Subject(s)
Nerve Agents , Rhodium , Rhodium/chemistry , Nerve Agents/analysis , Nerve Agents/chemistry , Coordination Complexes/chemistry , Organophosphorus Compounds/analysis , Organophosphorus Compounds/chemistry , Limit of Detection , Chromogenic Compounds/chemistry , Chemical Warfare Agents/analysis , Chemical Warfare Agents/chemistryABSTRACT
Regulation of the reaction pathways is a perennial theme in the field of chemistry. As a typical chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB) generally undertakes one-electron oxidation, but the product (TMBox1) is essentially a confused complex and is unstable, which significantly hampers the clinic chromogenic bioassays for more than 50 years. Herein, we report that sodium dodecyl sulfate (SDS)-based micelles could drive the direct two-electron oxidation of TMB to the final stable TMBox2. Rather than activation of H2O2 oxidant in the one-electron TMB oxidation by common natural peroxidase, activation of the TMB substrate by SDS micelles decoupled the thermodynamically favorable complex between TMBox2 with unreacted TMB, leading to an unusual direct two-electron oxidation pathway. Mechanism studies demonstrated that the complementary spatial and electrostatic isolation effects, caused by the confined hydrophobic cavities and negatively charged outer surfaces of SDS micelles, were crucial. Further cascading with glucose oxidase, as a proof-of-concept application, allowed glucose to be more reliably measured, even in a broader range of concentrations without any conventional strong acid termination.
Subject(s)
Hydrogen Peroxide , Micelles , Oxidation-Reduction , Peroxidase/metabolism , Benzidines/chemistry , Colorimetry , Chromogenic Compounds/chemistryABSTRACT
A series of [2-(nitroaryl)ethenyl]pyridinium and quinolinium derivatives have been synthesised as potential indicators of microbial nitroreductase activity. When assessed against a selection of 20 clinically important pathogenic microorganisms, microbial colonies of various colours (yellow, green, red, brown, black) were produced and attributed to nitroreductase activity. Most substrates elicited colour responses with Gram-negative microorganisms. In contrast, the growth of several species of Gram-positive microorganisms and yeasts was often inhibited by the substrates and hence coloured responses were not seen.
Subject(s)
Chromogenic Compounds , Nitroreductases , Chromogenic Compounds/chemistry , Substrate Specificity , Nitroreductases/metabolismABSTRACT
A paper microfluidic device capable of conducting enzyme-linked assays is presented: a microfluidic enzyme-linked paper analytical device (µEL-PAD). The system exploits a wash-free sandwich coupling to form beads/analyte/enzyme complexes, which are subsequently added to the vertical flow device composed of wax-printed paper, waxed nitrocellulose membrane and absorbent/barrier layers. The nitrocellulose retains the bead complexes without disrupting the flow, enabling for an efficient washing step. The entrapped complexes then interact with the chromogenic substrate stored on the detection paper, generating a color change on it, quantified with an open-source smartphone software. This is a universal paper-based technology suitable for high-sensitivity quantification of many analytes, such as proteins or nucleic acids, with different enzyme-linked formats. Here, the potential of the µEL-PAD is demonstrated to detect DNA from Staphylococcus epidermidis. After generation of isothermally amplified genomic DNA from bacteria, Biotin/FITC-labeled products were analyzed with the µEL-PAD, exploiting streptavidin-coated beads and antiFITC-horseradish peroxidase. The µEL-PAD achieved a limit of detection (LOD) and quantification <10 genome copies/µL, these being at least 70- and 1000-fold lower, respectively, than a traditional lateral flow assay (LFA) exploiting immobilized streptavidin and antiFITC-gold nanoparticles. It is envisaged that the device will be a good option for low-cost, simple, quantitative, and sensitive paper-based point-of-care testing.
Subject(s)
Chemistry Techniques, Analytical , Microfluidics , Paper , Microfluidics/instrumentation , Collodion/chemistry , Chromogenic Compounds/chemistry , Mobile Applications , Proteins/analysis , Nucleic Acids/analysis , Limit of Detection , Chemistry Techniques, Analytical/instrumentation , Chemistry Techniques, Analytical/methodsABSTRACT
The simple and sensitive detection of protein is of great significance in biological research and medical diagnosis. However, the commonly-used methods, such as enzyme-linked immunosorbent assay (ELISA), usually rely on signal tags labeled on the antibody, which limits the sensitivity and stability. Herein, we have designed and constructed a colorimetric immunosensor in this work for the analysis of protein by taking advantage of 2D metal-organic framework (2D-MOF) nanomaterials as enzyme mimics. The nanomaterial shows a strong peroxidase mimetic activity, and good selectivity after it is modified with a specific aptamer. Therefore, taking carcinoembryonic antigen (CEA) as an example, this immunosensor achieves a good detection performance with a linear range from 1 pg mL-1 to 1000 ng mL-1 and a limit of detection (LOD) of 0.742 pg mL-1. Moreover, the sensor can successfully distinguish the human serum of colorectal cancer patients from healthy people, which suggests that this sensor has great potential in clinical applications. More importantly, the mass production, low cost, stability and ease of transport of the MOFs nanomaterials, as well as the ability for visual detection will make this sensor suitable for point-of-care (POC) testing in remote or resource-poor areas.
Subject(s)
Carcinoembryonic Antigen/blood , Colorimetry/methods , Immunoassay/methods , Metal-Organic Frameworks/chemistry , Nanostructures/chemistry , Antibodies, Immobilized/immunology , Aptamers, Nucleotide/chemistry , Benzidines/chemistry , Biomarkers/blood , Carcinoembryonic Antigen/immunology , Catalysis , Chromogenic Compounds/chemistry , Colorectal Neoplasms/blood , Humans , Immobilized Nucleic Acids/chemistry , Limit of DetectionABSTRACT
Photoactive proteins typically rely on structural changes in a small chromophore to initiate a biological response. While these changes often involve isomerization as the "primary step", preceding this is an ultrafast relaxation of the molecular framework caused by the sudden change in electronic structure upon photoexcitation. Here, we capture this motion for an isolated model chromophore of the photoactive yellow protein using time-resolved photoelectron imaging. It occurs in <150 fs and is apparent from a spectral shift of â¼70 meV and a change in photoelectron anisotropy. Electronic structure calculations enable the quantitative assignment of the geometric and electronic structure changes to a planar intermediate from which the primary step can then proceed.
Subject(s)
Bacterial Proteins/chemistry , Chromogenic Compounds/chemistry , Coumaric Acids/chemistry , Photoreceptors, Microbial/chemistry , Chromogenic Compounds/radiation effects , Coumaric Acids/radiation effects , Isomerism , Light , Photochemical Processes/radiation effectsABSTRACT
Assays of pyrophosphate ion (PPi) are of remarkable biochemical significance due to their vital roles in the bioenergetic and metabolic processes or as disease indicators. Colorimetry is popular in the field of biosensing and detection because of its simplicity, speed and cost-effectiveness, but there is a lack of a suitable colorimetric probe. Herein, a novel colorimetric sensing platform has been established for the detection of pyrophosphate based on the FeMoO4-H2O2-3,3',5,5'-tetra-methylbenzidine (TMB) system. Compared with most previously reported iron-based nanozymes, the as-obtained FeMoO4 nanospheres with a rough surface possessed a much superior peroxidase-like catalytic activity (Vmax = 28.47 × 10-8 M s-1) and substrate affinity (Km = 0.174 mM) toward H2O2 catalysis. Due to the Fe(II) and PPi reaction, the presence of PPi could specifically restore blue oxidized TMB to colorless TMB, which led to a decrease in UV absorption at 652 nm. The absorbance change is proportional to the PPi concentration, with a linear detection range (from 0.5 to 25 µM) and a low detection limit of 0.3 µM (S/N = 3). Accordingly, its excellent selectivity and high sensitivity made it a potential colorimetric sensor for PPi analysis in actual water samples.
Subject(s)
Colorimetry/methods , Diphosphates/analysis , Nanospheres/chemistry , Benzidines/chemistry , Catalysis , Chromogenic Compounds/chemistry , Drinking Water/analysis , Hydrogen Peroxide/chemistry , Iron/chemistry , Limit of Detection , Molybdenum/chemistry , Oxidation-Reduction , Water Pollutants, Chemical/analysisABSTRACT
Novel Fe3O4@C@MnO2 composites were successfully synthesized for the first time via an interfacial reaction between magnetic porous carbon and KMnO4, in which the magnetic porous carbon was derived from the pyrolysis of Fe-MIL-88A under N2 atmosphere. Interestingly, the obtained Fe3O4@C@MnO2 composites were found to have triple-enzyme mimetic activity including peroxidase-like, catalase-like, and oxidase-like activity. As a peroxidase mimic, Fe3O4@C@MnO2 composites could catalyze the oxidation of TMB into a blue oxidized product by H2O2. As a catalase mimic, Fe3O4@C@MnO2 could catalyze the decomposition of H2O2 to generate O2 and H2O. As an oxidase mimic, Fe3O4@C@MnO2 could catalyze the direct oxidation of TMB to produce a blue oxidized product without H2O2. Reactive oxygen species measurements revealed that the oxidase-like activity originated from 1O2 and O2-âand littleâOH generated by the dissolved oxygen, which was catalyzed by the Fe3O4@C@MnO2 in the TMB oxidation reaction. The oxidase-like activity of Fe3O4@C@MnO2 was investigated in detail. Under the optimized conditions, a rapid, sensitive, visual colorimetric method for dopamine detection was developed based on the inhibitory effect of dopamine on the oxidase-like activity. The proposed method allows for dopamine detection with a limit of detection of 0.034 µM and a linear range of 0.125-10 µM. This new colorimetric method was successfully used for the determination of dopamine in human blood samples.
Subject(s)
Biosensing Techniques/methods , Carbon/chemistry , Dopamine/blood , Magnetite Nanoparticles/chemistry , Manganese Compounds/chemistry , Oxides/chemistry , Benzidines/chemistry , Benzothiazoles/chemistry , Catalysis , Chromogenic Compounds/chemistry , Colorimetry/methods , Dopamine/chemistry , Humans , Hydrogen Peroxide/chemistry , Limit of Detection , Metal-Organic Frameworks/chemistry , Oxidation-Reduction , Phenylenediamines/chemistry , Sulfonic Acids/chemistryABSTRACT
Mo5N6 nanosheets were synthesized by a nickel-induced growth method and were found to possess peroxidase-like activity in acidic condition and catalase-like activity in weak basic condition. In acidic condition, Mo5N6 nanosheets can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to form a blue color product (TMBOX). At the co-existence of 4-aminophenol (4-AP), 4-AP can react with H2O2 and TMBOX, resulting in the decrease of TMBOX and the fading of blue color. Therefore, a facile, sensitive colorimetric method for the quantitative detection of 4-AP was developed. The linear range for 4-AP was 1.0 to 80.0 µmolâ Lâ1 (R2 = 0.999), and the detection limit was 0.56 µmolâ Lâ1 based on 3σ/k. Resorcinol, aniline, humic acid, and common ions and anions in surface water did not interfere the determination of 4-AP. This colorimetric method was applied to measure the 4-AP in real water sample from Wulong River in Fujian Province of China. The relative standard deviation for the determination of 4-AP was ranged from 0.03 to 1.88%, and the recoveries from spiked samples were ranged between 99.2 and 107.6%. The determination results were consistent with those obtained by HPLC.
Subject(s)
Aminophenols/analysis , Colorimetry/methods , Nanostructures/chemistry , Water Pollutants/analysis , Aminophenols/chemistry , Benzidines/chemistry , Catalysis , Chromogenic Compounds/chemistry , Hydrogen Peroxide/chemistry , Limit of Detection , Oxidation-Reduction , Rivers/chemistry , Water Pollutants/chemistryABSTRACT
A novel colorimetric/fluorescent dual-mode nanosensor for Hg2+ detection was constructed using expanded mesoporous silica (EMSN)-encapsulated ultrasmall platinum nanoclusters (EMSN@Pt NCs) with improved peroxidase-like and stable fluorescent activities. The sensing technique was based on the mechanism that the peroxidase mimetic activity and fluorescence intensity of EMSN@Pt NCs can be inhibited in the presence of Hg2+. In this sensing platform, a linear range of 5-50 nM with a detection limit of 1.78±0.38 nM and quantification limit of 5.93 nM was obtained via fluorescent analysis. A linear calibration curve from 0.25 to 200 nM with a detection limit of 8.25±0.51 nM and quantification limit of 27.47 nM was achieved via colorimetric analysis. The proposed dual-mode probe possesses excellent selectivity and reliability for Hg2+ detection, which can function as an efficient nanosensor for the quantitative determination of Hg2+ in Pueraria lobata.
Subject(s)
Colorimetry/methods , Fluorescent Dyes/chemistry , Food Contamination/analysis , Mercury/analysis , Metal Nanoparticles/chemistry , Silicon Dioxide/chemistry , Benzidines/chemistry , Catalysis , Chromogenic Compounds/chemistry , Limit of Detection , Oxidation-Reduction , Platinum/chemistry , Porosity , Pueraria/chemistryABSTRACT
Recently, the design and development of nanozyme-based logic gates have received much attention. In this work, by engineering the stability of the nanozyme-catalyzed product, we demonstrated that the chromogenic system of 3, 3', 5, 5'-tetramethylbenzidine (TMB) can act as a visual output signal for constructing various Boolean logic operations. Specifically, cerium oxide or ferroferric oxide-based nanozymes can catalyze the oxidation of colorless TMB to a blue color product (oxTMB). The blue-colored solution of oxTMB could become colorless by some reductants, including the reduced transition state of glucose oxidase and xanthine oxidase. As a result, by combining biocatalytic reactions, the color change of oxTMB could be controlled logically. In our logic systems, glucose oxidase, ß-galactosidase, and xanthine oxidase acted as inputs, and the state of oxTMB solution was used as an output. The logic operation produced a colored solution as the readout signal, which was easily distinguished with the naked eye. More importantly, the study of such a decolorization process allows the transformation of previously designed AND and OR logic gates into NAND and NOR gates. We propose that this work may push forward the design of novel nanozyme-based biological gates and help us further understand complex physiological pathways in living systems.
Subject(s)
Benzidines/chemistry , Chemical Engineering/methods , Chromogenic Compounds/chemistry , Colorimetry/methods , Enzymes/chemistry , Nanotechnology , Biocatalysis , Catalysis , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Although organic artificial enzymes have been reported as biomimetic oxidation catalysts and are widely used for colorimetric biosensors, developing organic artificial enzymes with high enzymatic activity is still a challenge. Two-dimensional (2D) covalent organic frameworks (COFs) have shown superior potential in biocatalysts because of their periodic π-π arrays, tunable pore size and structure, large surface area, and thermal stability. The interconnection of electron acceptor and donor building blocks in the 2D conjugated COF skeleton can lead to narrower band gaps and efficient charge separation and transportation and thus is helpful to improve catalytic activity. Herein, a donor-acceptor 2D COF was synthesized using tetrakis(4-aminophenyl)pyrene (Py) as an electron donor and thieno[3,2-b]thiophene-2,5-dicarbaldehyde (TT) as an electron acceptor. Under visible light irradiation, the donor-acceptor 2D COF exhibited superior enzymatic catalytic activity, which could catalyze the oxidation of chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) by the formation of superoxide radicals and holes. Based on the above property, the photoactivated donor-acceptor 2D COF with enzyme-like catalytic properties was designed as a robust colorimetric probe for cheap, highly sensitive, and rapid colorimetric detection of glutathione (GSH); the corresponding linear range of GSH was 0.4-60 µM, and the limit of detection was 0.225 µM. This study not only presents the construction of COF-based light-activated nanozymes for environmentally friendly colorimetric detection of GSH but also provides a smart strategy for improving nanozyme activity.
Subject(s)
Glutathione/blood , Metal-Organic Frameworks/chemistry , Nanostructures/chemistry , Benzidines/chemistry , Catalysis/radiation effects , Chromogenic Compounds/chemistry , Colorimetry/methods , Density Functional Theory , Glutathione/chemistry , Humans , Light , Limit of Detection , Metal-Organic Frameworks/chemical synthesis , Metal-Organic Frameworks/radiation effects , Models, Chemical , Nanostructures/radiation effects , Oxidation-Reduction , Pyrenes/chemical synthesis , Pyrenes/chemistry , Pyrenes/radiation effects , Thiophenes/chemical synthesis , Thiophenes/chemistry , Thiophenes/radiation effectsABSTRACT
Pressure-based immunoassays have been studied for point-of-care testing for which increasing the sensitivity is still a challenge. In this study, we described an enhanced pressure-based immunoassay with a versatile electronic sensor for the sensitive biological analysis. The versatile electronic sensor had multifunctional sensing capabilities with temperature and pressure recording. Magnetic bead-modified capture antibody and platinum nanoparticle-labeled detection antibody were used as the biorecognition element of the target carcinoembryonic antigen (CEA) (as a model analyte) and would form a sandwich-type immune complex with CEA. After simple magnetic separation, this complex was transferred into the detection chamber, which contained both hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). With the catalytic ability of PtNPs, the "H2O2-TMB-PtNPs" system was catalyzed to generate a large amount of oxygen (O2) and photothermal agent of oxidizer TMB (ox-TMB). Meanwhile, in a sealed chamber, further irradiation with an 808 nm near-infrared laser led to a triple-step signal amplification strategy of pressure increase, temperature increase, and gas thermal expansion to receive a strong electrical signal through the electronic sensor in real time. Thus, the amplified electrical signal from the electronic sensor could reveal the target concentration. In addition, we also verified that the synergistic system with two physical quantities had a lower limit of detection and a wider detection range compared to the detection system with a single physical quantity. In general, this immunoassay not only helped in exploring an effective signal amplification pathway but also offered an opportunity for the development of versatile electronic sensors in point-of-care settings.
Subject(s)
Carcinoembryonic Antigen/blood , Electrochemical Techniques/methods , Immunoassay/methods , Pressure , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Benzidines/chemistry , Carcinoembryonic Antigen/immunology , Chromogenic Compounds/chemistry , Electrochemical Techniques/instrumentation , Graphite/chemistry , Humans , Hydrogen Peroxide/chemistry , Immunoassay/instrumentation , Limit of Detection , Metal Nanoparticles/chemistry , Platinum/chemistry , Proof of Concept Study , TemperatureABSTRACT
A practical colorimetric assay was developed for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For this purpose, magnetic γ Fe2O3 nanoparticles were synthesized and used as a peroxidase-like mimic activity molecule. In the presence of γ Fe2O3 nanoparticles, the color change of H2O2 included 3,3',5,5'-tetramethylbenzidine was monitored at the wavelength of 654 nm when spike protein interacted with angiotensin-converting enzyme 2 receptor. This oxidation-reduction reaction was examined both spectroscopically and by using electrochemical techniques. The experimental parameters were optimized and the analytical characteristics investigated. The developed assay was applied to real SARS-CoV-2 samples, and very good results that were in accordance with the real time polymerase chain reaction were obtained.
Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Colorimetry/methods , Magnetic Iron Oxide Nanoparticles/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Benzidines/chemistry , Biosensing Techniques/methods , COVID-19 Testing/instrumentation , Catalysis , Chromogenic Compounds/chemistry , Cysteine/chemistry , Humans , Hydrogen Peroxide/chemistry , Limit of Detection , Nasopharynx/virology , Oropharynx/virology , Oxidation-Reduction , Peroxidase/chemistry , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
A comparative analysis of the performance of the new selective chromogenic CHROMagar™-Serratia culture medium for detection and isolation of Serratia marcescens was undertaken. A total of 134 clinical isolates (95 S. marcescens with and without carbapenemase production and 39 non-S. marcescens isolates) and 96 epidemiological samples (46 rectal swabs and 50 from environmental surfaces) were studied. Diagnostic values when compared with CHROMagar™-Orientation medium were 96.8% sensitivity, 100% specificity, 100% positive predictive value and 88.5% negative predictive value. In conclusion, CHROMagar™-Serratia shows an excellent ability for differentiation of S. marcescens among clinical isolates and in environmental samples.
Subject(s)
Bacteriological Techniques/methods , Culture Media/chemistry , Serratia Infections/microbiology , Serratia marcescens/growth & development , Serratia marcescens/isolation & purification , Agar/chemistry , Agar/metabolism , Bacteriological Techniques/instrumentation , Chromogenic Compounds/chemistry , Chromogenic Compounds/metabolism , Culture Media/metabolism , Humans , Serratia Infections/diagnosis , Serratia marcescens/metabolismABSTRACT
A colorimetric and fluorescent dual-channel detection method for acid phosphatase (ACP) activity has been constructed, based on the internal filtering effect between oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) and rhodamine B (RB). Au3+, which in situ form gold nanoparticles (AuNPs), can oxidize colorless 3,3',5,5'-tetramethylbenzidine (TMB) to oxTMB (blue color). The fluorescence of RB can be quenched by oxTMB due to the spectral overlap of emission of RB and absorption of oxTMB. By means of the above process, ACP can be determined because ACP promotes the hydrolysis of 2-phospho-L-ascorbic acid trisodium salt (AAP) to generate ascorbic acid (AA), which can inhibit the internal filtering effect between RB and oxTMB. No material preparation was needed for the determination of ACP. The colorimetric and fluorimetric methods can quantify ACP in the range 0.06-5.0 mU/mL and 0.03-5.0 mU/mL, respectively. Furthermore, a smartphone-assisted sensing platform has been constructed for on-site monitoring of ACP in the range 0.75-50 mU/mL, and the detection limit is 0.3 mU/mL. The methods developed can measure ACP in human serum successfully.
Subject(s)
Acid Phosphatase/blood , Colorimetry/methods , Spectrometry, Fluorescence/methods , Acid Phosphatase/chemistry , Ascorbic Acid/analogs & derivatives , Ascorbic Acid/chemistry , Benzidines/chemistry , Chlorides/chemistry , Chromogenic Compounds/chemistry , Colorimetry/instrumentation , Fluorescent Dyes/chemistry , Gold Compounds/chemistry , Humans , Limit of Detection , Oxidation-Reduction , Rhodamines/chemistry , Smartphone , Spectrometry, Fluorescence/instrumentationABSTRACT
In this work, we fabricated a novel origami paper-based analytical device (oPAD) assisted by the nanostructuring effect of in situ Pd decoration of Cu/Co-doped CeO2 (CuCo-CeO2-Pd) nanospheres, functionalized with their strongly enhanced electrocatalytic properties to realize an electrochemical and visual signal readout system in oPAD, for highly sensitive detection of amyloid-ß (Aß). The CuCo-CeO2-Pd nanospheres were introduced as an enhanced "signal transducer layer" on account of the electron transfer acceleration caused by catalyzing glucose to produce H2O2 for differential pulse voltammetry signal readout and further 3,3'5,5'-tetramethylbenzidine (TMB) oxidation for colorimetric analysis. Meanwhile, for achieving superior performance of the proposed oPAD, in situ growth of urchin-like gold nanoparticles (Au NPs) onto cellulose fibers was adopted to improve "the recognition layer" in favor of immobilizing antibodies for targeting Aß through specific antigen-antibody interactions. Combined with the delicate design of oPAD, exhibiting actuation of the conversion procedure between hydrophobicity and hydrophilicity on paper tabs in the assay process, the oPAD successfully enabled sensitive diagnosis of Aß in a linear range from 1.0 pM to 100 nM with a limit of detection of 0.05 pM (S/N = 3) for electrochemical detection, providing a reliable strategy for quantifying the Aß protein in clinical applications.
Subject(s)
Amyloid beta-Peptides/analysis , Electrochemical Techniques/instrumentation , Metal Nanoparticles/chemistry , Paper , Amyloid beta-Peptides/blood , Amyloid beta-Peptides/cerebrospinal fluid , Benzidines/chemistry , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Catalysis , Cerium/chemistry , Chromogenic Compounds/chemistry , Electrochemical Techniques/methods , Glucose Oxidase/chemistry , Gold/chemistry , Limit of Detection , Nanospheres/chemistry , Oxidation-Reduction , Palladium/chemistry , Reproducibility of ResultsABSTRACT
Chromogenic immunohistochemistry (immunostaining using an enzyme-labeled probe) is an essential histochemical technique for analyzing pathogenesis and making a histopathological diagnosis in routine pathology services. In neoplastic lesions, immunohistochemistry allows the study of specific clinical and biological features such as histogenesis, behavioral characteristics, therapeutic targets, and prognostic biomarkers. The needs for appropriate and reproducible methods of immunostaining are prompted by technical development and refinement, commercial availability of a variety of antibodies, advanced applicability of immunohistochemical markers, accelerated analysis of clinicopathological correlations, progress in molecular targeted therapy, and the expectation of advanced histopathological diagnosis. However, immunostaining does have various pitfalls and caveats. Pathologists should learn from previous mistakes and failures and from results indicating false positivity and false negativity. The present review article describes various devices, technical hints, and trouble-shooting guides to keep in mind when performing immunostaining.