Electrochemical and colorimetric dual-signal detection of Staphylococcus aureus enterotoxin B based on AuPt bimetallic nanoparticles loaded Fe-N-C single atom nanocomposite

Sensitive detection of Staphylococcus aureus enterotoxin B(SEB)is of importance for preventing food poisoning from threatening human health.In this work,an electrochemical and colorimetric dual-signal detection assay of SEB was developed.The probe(Ab2/AuPt@Fe-N-C)was bound to SEB captured by Ab1,where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal.Furthermore,the probe catalyzed the oxidation of 3,3’,5,5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm.Once the target was captured and formed a sandwich-like complex,the color changed from colorless to blue.SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002-10.0000 and 0.0005-10.0000 ng/mL,with limits of detection of 0.0667 and 0.1670 pg/mL,respectively.The dual-signal biosensor was successfully used to detect SEB in milk and water samples,which has great potential in toxin detection in food and the environment.

One stone two birds:electrochemical and colorimetric dual-mode biosensor based on copper peroxide/covalent organic framework nanocomposite for ultrasensentive norovirus detection

Norovirus(NoV)is regarded as one of the most common causes of foodborne diarrhea in the world.It is urgent to identify the pathogenic microorganism of the diarrhea in short time.In this work,we developed an electrochemical and colorimetric dual-mode detection for NoV based on the excellent dual catalytic properties of copper peroxide/COF-NH_(2)nanocomposite(CuO_(2)@COF-NH_(2)).For the colorimetric detection,NoV can be directly detected by the naked eye based on CuO_(2)@COF-NH_(2)as a laccase-like nonazyme using“peptide-NoV-antibody”recognition mode.The colorimetric assay displayed a wide and quality linear detection range from 1 copy/mL to 5000 copies/mL of NoV with a low limit of detection(LOD)of 0.125 copy/mL.For the electrochemical detection of NoV,CuO_(2)@COF-NH_(2)showed an oxidation peak of copper ion from Cu^(+)to Cu^(2+)using“peptide-NoV-antibody”recognition mode.The electrochemical assay showed a linear detection range was 1-5000 copies/mL with a LOD of 0.152 copy/mL.It's worthy to note that this assay does not need other electrical signal molecule,which provide the stable and sensitive electrochemial detection for NoV.The electrochemical and colorimetric dual-mode detection was used to detect NoV in foods and faceal samples,which has the potential for improving food safety and diagnosing of NoV-infected diarrhea.

Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).

Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles（GNPs） based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay. In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy（CLSM） and classic flow cytometry（FCM） assay, and satisfactory results were obtained.

Gold Nanoparticle-based Colorimetric Assay for Determination of Lead(Ⅱ) in Aqueous Media

The authors presented a simple colorimetric assay for the detection of toxic heavy metal lead（Ⅱ） ion（Pb2＋） Pentapeptide, cysteine-alanine-leacine-asparagine-asparagine（CALNN）, functionalized gold nanoparticles（GNPs） were aggregated in the presence of the divalent metal ion in solution by an ion-templated chelation process, which caused an easily measurable change in the absorption spectrum of the particles. Typically, mono-dispersing GNPs exhibit an absorption band at 522 nm, corresponding to a red color solution, while aggregated GNPs have it at longer wavelengths, corresponding to a purple or blue color solution. The chelation/aggregation process is reversible via the addition of a strong metal ion chelator such as EDTA. Highly selective and sensitive detection of Pb^2＋ in aqueous solution is thus provided. A detection limit of 0.1 μmol/L of Pb^2＋ was demonstrated.

New Colorimetric Method for Lipases Activity Assay in Microbial Media

A simple, rapid and precise method has been developed for determination of lipase activity in microbial media. The method is based on using phenyl acetate as substrate for lipase and determination of liberated phenol by Folin Ciocalteu reagent. Reaction mixture containing substrate 2.4 ml of phenyl acetate 165 μM in Tris HCl buffer, 0.1 M and pH 7, with 1% (v/v) Triton X-100) and 0.1 ml lipase is incubated at 40?C during 10 minutes and the absorbance was measured at 750 nm. Linearity was observed in the concentration range 0-0.8 g/L lipase.

Development of a Label-Free Colorimetric Aptasensor with Rationally Utilized Aptamer for Rapid Detection of Okadaic Acid

Okadaic acid(OA)is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning(DSP).Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analysis.In this study,a label-free col-orimetric aptasensor was constructed for visual and rapid detection of OA in shellfish.To exploit the binding capability of the anti-OA aptamer,the inherent molecular recognition mechanism of aptamer and OA was studied,based on molecular docking,fluorescent assay,and biolayer interferometry.Consistent results showed that the stem-loop near the 3’terminal of the aptamer exhibit dominate binding capacity.Based on the revealed recognition information,the aptamer was thus rationally utilized and combined with AuNPs and cationic polymer polydiallyl dimethyl ammonium chloride(PDDA)for the development of the label-free colorimetric aptasensor,in which the 3’terminal was thoroughly exposed to OA.The aptasensor provided robust performance with a linear detection range of 100-1200 nmol L-1,a limit of detection of 41.30 nmol L-1,recovery rates of 91.6%-106.2%,as well as a high selectivity towards OA in shellfish samples.The whole detection process can be completed within 1 h.To our best knowledge,this is the first time that the anti-OA aptamer was thoroughly studied,and a label-free colorimetric aptasensor was rationally designed in this way.This study not only provides a rapid detection method for highly sensitive and specific detection of OA,but also serves as a reference for the design of efficient aptasensors in the future.

Polydopamine-Based Colorimetric Superwettable Biosensor for Highly Sensitive Detection of Hydrogen Peroxide and Glucose

Superwettable surface has broad application prospects in fabricating biosensors due to its significant enrichment effect.Here,we report a polydopamine-based colorimetric superwettable sensor that integrates superhydrophobic-superhydrophilic micropatterns for the determination of hydrogen peroxide(H_(2)O_(2))and glucose.Dopamine can be oxidized into polydopamine with the addition of horseradish peroxidase(HRP)and H_(2)O_(2),leading to the deposited spots color change from colorless to black.The concentration of target can be determined by analyzing RGB value using a smartphone software.The superhydrophobic area on the superwettable surface helps capture droplets by confining them to superhydrophilic microwells.After droplet evaporation,the analytes are concentrated in the small superhydrophilic domain,thus greatly enhancing the sensitivity.The experimental results manifested that superwettable sensor is able to detect H_(2)O_(2)with a broad linear range of 0.25μmol/L-25 mmol/L and a low limit of detection(LOD)of 0.25μmol/L by naked eye.For glucose detection,the linear range of the sensor is from 2μmol/L to 20 mmol/L and LOD is 0.69μmol/L.The superwettable sensor has been successfully applied in practical samples,including cancerous cells,milk,urine,and human serum samples with acceptable results.This superwettable sensor has several merits,such as high sensitivity,rapid response,and low sample volume in a single microdroplet,and shows great potential in manufacturing portable devices for complex biosensing applications.

Target-induced mimic enzyme deactivation based on mixed-node metal-organic frameworks for colorimetric assay of hydrogen sulfide

Accurate detection of hydrogen sulfide(H_(2)S)is of great significance for environmental monitoring and protection.We propose a colorimetric method for the detection of H_(2)S by the use of mixed-node Cu-Fe metal organic frameworks(Cu-Fe MOFs)as highly efficient mimic enzymes for target-induced deactivation.The Cu-Fe MOFs were synthesized by a simple solvothermal method and could catalyze the H_(2)O_(2)mediated oxidation of 3,30,5,50-tetramethylbenzidine(TMB)to oxTMB with a blue color.The presence of dissolved H_(2)S would deactivate the mimic enzymes,and then the blue color disappeared.The mechanism of the sensor was discussed by steady-state kinetic analysis.The designed assay was highly sensitive for H_(2)S detection with a linear range of 0à80 mmol/L and a detection limit of 1.6 mmol/L.Moreover,some potential substances in the water samples had no interference.This method with the advantages of low cost,high sensitivity,selectivity,and visual readout with the naked eye was successfully applied to the determination of H_(2)S in industrial wastewater samples.

Colorimetric Assay to Determine In Vitro Antibacterial Activity Against Clinical Isolates： Enhanced Activity in Damaged Chinese Masson Pine Needles

A colorimetric assay for antibacterial susceptibility testing of clinical isolates （Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Bacillus cereus, and Streptococcus pneumoniae） is described based on the reduction of a novel tetrazolium salt, 3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2Htetrazolium （MTS）, in the presence of phenazine methosulfate （PMS） as an electron-coupling agent. The combination of 200 μg/mL MTS with 25 μmol/L PMS resulted in production of large amounts of formazan within 1 h of exposure. In this setting, fractions extracted from Chinese Masson pine （Pinus massoniana Lamb.） needles damaged by the pine caterpillar Dendrolimus punctatus Walker were found to have enhanced levels of antibacterial activity. These fractions, which were designated ＂Master＂, ＂Technique＂, and ＂Strength＂, were isolated and identified by reverse-phase C18 cartridge concentration, gel filtration, and affinity chromatography. Two fractions purified from healthy and undamaged needles were designated H1 and H2, respectively. For all test bacteria species. Technique produced the lowest minimal inhibitory concentrations （MICs）, ranging from 2 to 32 μg/mL, and H2 produced the highest values, with four of the six M ICs being higher than 128 μg/mL. We found that the Rmax model fitted the data well in that the r^2 ranged between 0.87 and 0.96 （median, 0.92） and no statistically significant deviations from the model were found （P= 0.23）. The median coefficient of variation of the log RC50 values and the slope m of the fitted model for all six strains among the replicates were 38 and 41%, respectively. In the course of the investigation, the physiological and functional factors involved in pest damage to plants were also explored. In summary, the MTS-PMS colorimetric assay has advantages over existing methods for the examination of antibacterial activity, and could be developed further such that it would be suitable for screening new antibiotic molecules.

A quantitative colorimetric assay of H_2O_2 and glucose using silver nanoparticles induced by H_2O_2 and UV

A simple spectrophotometric assay of H2O2 and glucose using Ag nanoparticles has been carried out. Relying on the synergistic effect of H2O2 reduction and ultraviolet （UV） irradiation, Ag nanoparticles with enhanced absorption signals were synthesized. H2O2 served as a reducing agent in the Ag nanoparticles formation in which Ag＋ was reduced to Ago by O2- generated via the decomposition of H2O2 in alkaline media. On the other hand, photoreduction of Ag＋ to Ago under UV irradiations also contributed to the nanoparticles formation. The synthesized nanoparticles were characterized by TEM, XPS, and XRD. The proposed method could determine H2O2 with concentrations ranging from 5.0× 10^-7 to 6.0× 10^-5 tool/ L The detection limit was estimated to be 2.0 × 10^-7 mol/L. Since the conversion of glucose to gluconic acid catalyzed by glucose oxidase was companied with the formation of H2O2, the sensing protocol has been successfully utilized for the determination of glucose in human blood samples. The results were in good agreement with those determined by a local hospital. This colorimetric sensor thus holds great promises in clinical applications.

Colorimetric detection of glucose using a boronic acid derivative receptor attached to unmodified AuNPs

A simple, cheap and non-enzymatic colorimetric strategy for glucose detection has been designed based on the interactions between a phenylboronic acid （PBA） derivative, which is coupled with gold nanoparticles （AuNPs） as the colorimetric reporters, and glucose. The PBA-AuNPs hybrid system proposed here exhibits ordered photochemistry behaviors upon the addition of glucose at different pH values. There are two linear regions of glucose concentration for the glucose sensor at different pH values, i.e., between 0.1 mmol/L and 9.8 mmol/L at pH 6 with the detection limit of 64μmol/L and between 0 and 6.5 mmol/L with the detection limit of 48 μmol/L at pH 9, respectively. To test the practicality of the sensor system, we also applied this assay to detect a glucose sample in the artificial saliva.

Visual detection of tropomyosin,a major shrimp allergenic protein using gold nanoparticles(AuNPs)-assisted colorimetric aptasensor

A gold nanoparticle-based label-free colorimetric assay was developed to detect the shrimp allergenic protein tropomyosin(TM),an important biomarker responsible for severe clinical reactivity to shellfish.In a gold nanoparticles(AuNPs)-tropomyosin-binding aptamer(TMBA)complex,the aptamer adsorbs onto the surface of AuNPs and dissociates in the presence of TM.In addition,AuNPs tend to aggregate in the presence of ionic salt,revealing a color change(i.e.,wine-red to purple/blue)with a shift in the maximum absorption peak from 520 nm.In the presence of specific binding TM,the aptamer folds into a tertiary structure where it more efficiently stabilizes AuNPs toward the salt-induced aggregation with a hypsochromic shift in the absorption spectra compared to the stabilized AuNPs by aptamer alone.Based on the aggregation and sensitive spectral transformation principle,the AuNPs-based colorimetric aptasensor was successfully applied to detect TM with a range of 10-200 nmol/L and a low detection limit of 40 nmol/L in water samples.The reliability,selectivity,and sensitivity of the aptasensor was then tested with food samples spiked with TM.The observed detection limit was as low as 70 nmol/L in shrimp,90 nmol/L in tofu,and 80 nmol/L in eggs,respectively.We anticipate the proposed AuNPs-based colorimetric aptasensor assay possesses a high potential for the easy and efficient visual colorimetric detection of TM.

AuNP aggregation-induced quantitative colorimetric aptasensing of sulfadimethoxine with a smartphone

A gold nanoparticle(AuNP)aggregation-induced colorimetric aptasensing method for quantitative detection of sulfadimethoxine(SDM)with a smartphone was developed.AuNPs were complexed with aptamers which protected Au NPs from aggregating in high-concentration salt solutions.In the presence of SDM,SDM bound with the aptamer on the surface of Au NPs with higher affinity,which competitively desorbed the aptamer from the AuNP surface and resulted in AuNPs aggregation,accompanied with a color change from red to purple-blue.The R,G and B values of images taken by a smartphone camera were analyzed with an app on the smartphone,and were utilized for quantitative analysis of SDM.Under the optimized conditions,the colorimetric aptasensing method using a smartphone showed high sensitivity for SDM,with the limit of detection of 0.023 ppm,lower than the allowed maximum SDM residue limit.This study provides a simple,fast,and easy to read method for on-site quantitative biochemical and cellular analysis.

Colorimetric and electrochemical detection of ligase through ligation reaction-induced streptavidin assembly

We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin(SA)-biotin interaction.Sortase A(Srt A)served as the model analyte and two biotinylated peptides(bio-LPETGG and GGGK-bio)were used as the substrates.Srt A-catalyzed ligation of the peptide substrates led to the generation of bio-LPETGGGKbio.The ligation product(bio-LPETGGGK-bio)induced the aggregation and color change of SA-modified gold nanoparticles(Au NPs)through the SA-biotin interactions,which could be assayed by the colorimetric method.Furthermore,we found that the bio-LPETGGGK-bio could trigger the assembly of tetrameric SA proteins with the formation of the(SA-bio-LPETGGGK-bio)nassemblies through the same interactions.The above results were further confirmed by atomic force microscopy and fluorescent imaging.The insulated assemblies were in-situ fabricated at the SA-modified gold electrode,thus hindering the electron transfer of[Fe(CN)_(6)]^(3-/4-) and leading to an increase in the electron-transfer resistance.The capability of the method for the detection of Srt A both in vitro and Staphylococcus aureus(S.aureus)has been demonstrated.Srt A with a concentration down to 1 pmol/L has been determined by the electrochemical analysis,which is lower than that achieved by the colorimetric assay(50 pmol/L).By integrating the advantages of homogeneous reaction and heterogeneous detection,the strategy serves as an ideal means for the fabrication of various sensing platforms by adopting biotin-labeled and sequence-specific peptide or nucleic acid substrates.

Verification and Improvement of the MTT Method for in vitro HSS Bioactivity Activity Determination

To optimize the experimental conditions of MTT colorimetric assay for HSS bioactivity in vitro,we studied the optimal combination of the major conditions of the MTT assay by orthogonal test and other experiments,and compared HSS bioactivity in vitro measured by the improved MTT protocol and published MTT assay at serial protein doses.Results showed that the absorbance value(A value)of the MTT assay directly correlated with the number of human hepatoma cell lines SMMC7721.The result of orthogonal test was the number of 5×104 SMMC7721 cells/ml,culture period 6 h before adding HSS,concentration of HSS 100μg/ml,incubation time with HSS 36 h.Additionally,several experiments demonstrated the optimal combination of other conditions was 50μg MTT,incubation time for MTT 6 h,DMSO was used to dissolve the MTT formazan crystals and measured with ELISA scanner at 570 nm.The result of determining HSS bio-activity in vitro by optimized MTT protocol showed that sHSS bio-activity increased with the growth of protein dose,but decreased when it beyond a certain dose.The optimized MTT protocol was a sensitive,convenient and stable quantitative method to evaluate HSS bio-activity.

Bimetallic Fe-Co Nanoalloy Confined in Porous Carbon Skeleton with Enhanced Peroxidase Mimetic Activity for Multiple Biomarkers Monitoring

Carbon-based nanozymes with multifunctional applications have attracted enormous attention,however,there is still a lack of an effective strategy for inexpensive preparation of high-active carbon-based nanozyme.We herein report a waste paperderived CoFe_(2)O_(4)/porous carbon nanozyme for the colorimetric detection of glucose and glutathione(GSH)in biofluids.The hybrid CoFe_(2)O_(4)/porous carbon material was successfully prepared via a combined impregnation,hydrolysis and carbonization method.Newly constructed CoFe_(2)O_(4)/porous carbon material with enhanced peroxidase-like activity could oxidize colorless tetramethylbenzidine(TMB)to blue oxidized TMB with adding H_(2)O_(2).A sensitive colorimetric analysis platform for monitoring the levels of glucose and GSH in biofluids was respectively developed.The proposed analytical method possessed predominant features such as low limit of detections(LODs)(0.16μmol/L for glucose and 120 nmol/L for GSH),broad linear ranges(1-200μmol/L for glucose and 1-50μmol/L for GSH),and excellent practical potential.This multipurpose platform not only provides a promising strategy for transforming waste paper into valuable carbon-based nanozyme,but also paves the way for excavating the potential biomedical and environmental applications from waste paper.

Intelligent droplet tracking with correlation filters for digital microfluidics

Tracking the movement of droplets in digital microfluidics is essential to improve its control stability and obtain dynamic information for its applications such as point-of-care testing,environment monitoring and chemical synthesis.Herein,an intelligent,accurate and fast droplet tracking method based on machine vision is developed for applications of digital microfluidics.To continuously recognize the transparent droplets in real-time and avoid the interferes from background patterns or inhomogeneous illumination,we introduced the correlation filter tracker,enabling online learning of the multi-features of the droplets in Fourier domain.Results show the proposed droplet tracking method could accurately locate the droplets.We also demonstrated the capacity of the proposed method for estimation of the droplet velocity as faster as 20 mm/s,and its application in online monitoring the Griess reaction for both colorimetric assay of nitrite and study of reaction kinetics.

A novel efficient medium for chromogenic catalysis of tetramethylbenzidine with horseradish peroxidase

The traditional surfactant sodium dodecyl sulfate（SDS） and ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate（[Emim][BF4]） have been combined to create a novel efficient medium for chromogenic catalysis of 3,30,5,50-tetramethylbenzidine with horseradish peroxidase in presence of H2O2. The results have shown the [Emim][BF4] in the mediums can promote the rate of formation of the blue chromogen,the SDS is responsible for the stabilization of the blue chromogen due to the electrostatic attraction between positively charged blue chromogen and the negatively charged surfactant. The SDS/[Emim][BF4]combination not only enhance catalytic activity of HRP remarkably but also stabilize the blue chromogen formed in the HRP oxidation of the substrate TMB compared to the conventional medium. Based on the superior combination of SDS and [Emim][BF4], the colorimetric assay for detecting HRP activity and H2O2 concentration was established. This work demonstrates a novel efficient medium for chromogenic catalysis with potential applications in biosensors and clinical diagnosis.