Streptavidin-horseradish Peroxidase Research Articles

Development of a simple enzyme-linked hybrid-sandwich assay for sensitive detection of cardiac troponin I

This work reports a simple enzyme-linked hybrid-sandwich assay (ELHSA) for one vital cardiac biomarker, cardiac troponin I (cTnl). Aimed at expanding the “antibody-antigen” immunoassay, a distinct “capture antibody-target-detection aptamer” configuration was designed, in which two types of recognition elements were involved. Through systematical study, optimal antibody-aptamer combination and amount ratio between streptavidin-horseradish peroxidase conjugate and biotin-modified aptamer were obtained, and a beneficial interaction mode between cTnI and the two recognition elements was disclosed, namely, cTnI protein binding to the aptamer could be well recognized by antibody, but not vice versa. Moreover, it was first found that, apart from as a blocking agent, bovine serum albumin could be introduced in the detection probe solution as an enhancer, leading to high sensitivity and reproducibility. The BSA-assisted ELHSA can detect as low as 0.24 ng/mL cTnI and is a new, simple and effective strategy to apparently improve ELHSA for cTnI detection. Meanwhile, prominent selectivity derived from dual specific recognition elements and good applicability indicate that the developed ELHSA for cTnI is anticipated to substitute classical ELISA for diagnosis of cardiovascular diseases in the future.

Supersensitive detection of single-histamine molecule on nanoplates by turn-on small molecule fluorescence sandwich immunoassay.

We develop a supersensitive "turn-on format" fluorescence sandwich immunoassay for detecting small single molecules. Gold nanoplate-based biotin antibodies and streptavidin-fluorophores were used instead of streptavidin-horseradish peroxidase reacting with a biotin tracer in a microplate-based competitive enzyme-linked immunosorbent assay (ELISA). Our platform showed a low detection limit of 5 zeptomolar (5 × 10-21 M), 5.4 × 1010 times higher detection sensitivity than the conventional tune-off format ELISA.

An electrochemical paper-based hydrogel immunosensor to monitor serum cytokine for predicting the severity of COVID-19 patients

Analysis of cytokines levels in human serum is critical as it can be a “symptom diagnostic biomarker” in COVID-19, giving real-time information about human health status. Here, we present the construction and performance of a low-price immunosensor (∼US$0.428 per test) based on microfluidic paper-based system to detect cytokine for predicting the health status of COVID-19 patients. Interleukin-6 (IL-6) was selected as the detection model for the close relationship between IL-6 and COVID-19. The assay, which we integrated into foldable paper system, leverages the magnetic immunoassay, the streptavidin-horseradish peroxidase (HRP) associated with tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) to amplify the signal for electrochemical readout. To improve the sensitivity of cytokine detection, a hybrid of gold nanoparticles (AuNPs) and polypyrrole (PPy) hydrogel was modified on the working electrode to increase the conductivity and improve the electron transfer rate. With our prototypic origami paper-based immunosensor operated in differential pulse voltammetry (DPV) mode, we achieved excellent results with a dynamic range from 5 to 1000 pg/mL and a lower detection limit (LOD) of 0.654 pg/mL. Furthermore, we evaluated the capability of the clinical application of the proposed immunosensor using human serum samples from a hospital. The results indicate that our proposed immunosensor has great potential in early diagnosing high-risk COVID-19 patients.

Preparation of monoclonal antibodies against Pinellia ternata lectin protein and establishment of double-antibody sandwich ELISA

To determine the content of endogenous toxic substance Pinellia ternata lectin(PTL) protein in Pinelliae Rhizoma and the related processed products, this study prepared specific monoclonal antibodies against PTL by hybridoma cell technology, and established a quantitative double-antibody sandwich enzyme linked immunosorbent assay(ELISA) for PTL antigen. The detection conditions were 2.5 μg·mL~(-1) working concentration of the captured antibody and 1∶450 of the dilution multiple of detected antibody. The coating condition was staying overnight at 4 ℃. The blocking time and incubation times of antigen and detected antibody were all 90 minutes. The incubation time of horseradish peroxidase conjugated streptavidin-horseradish peroxidase(SA-HRP) was 15 minutes. The quantitative limit of the method for PTL antigen was 0.375 ng·mL~(-1). The linear range was 75.000-4 800.000 pg·mL~(-1), and R~2=0.997 1. The recovery rate was 90.0%-110.0%, and the variation coefficients of intra-test and inter-test precision were 2.0%-3.0% and 2.0%-8.5%.The content of PTL in three batches of Pinelliae Rhizoma and the related processed products was determined by the method, and the average content of PTL in Pinelliae Rhizoma was 35.42 mg·g~(-1). The average content of PTL in Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine were 1.15 mg·g~(-1), 16.53 μg·g~(-1), and 122.63 ng·g~(-1), respectively, indicating that the content of PTL decreased significantly after processing. The quantitative double-antibody sandwich ELISA for PTL antigen established in this paper had good linearity, sensitive response, and high accuracy, which provided a simple and effective monitoring method for the detection of PTL content in the processing of Pinelliae Rhizoma.

Ultra-Low Detection of Infectious Disease Nucleic Acid Biomarkers By a Visual Colorimetric Sensor

The gold standard method for detecting nucleic acid markers is the polymerase chain reaction. However, this method demands high technical skills, labor-intensive, time-consuming, and expensive. Many resource-limited countries around the globe do not have access to such laboratory-based PCR tests. To address the global test needs of virus and other pathogen infections, as well as address the early biomarker detection of diseases. We have developed an affordable nanoparticle colorimetric sensor approach. In our system, the virus RNA marker is captured by the complementary oligonucleotide attached to low fouling gold-coated iron oxide nanoparticles. Further specificity is achieved through a second complementary hybridization probe conjugated with streptavidin-horseradish peroxidase labels. Visual colorimetry is performed using a chromogenic substrate allowing the visual conversion of the colorless solution to a blue color confirming a positive test. The intensity of the color is in direct correlation to the concentration of the virus RNA marker. We have successfully demonstrated attomolar detection in buffer and picomolar detection in real undiluted biofluids such as in neat human serum. Our ongoing efforts focus on saliva-based non-invasive detection with ultra-low capabilities, optimization of the assay, repeatability, and expanding this work for a multiplex detection format

Biomanufacturing Biotinylated Magnetic Nanomaterial via Construction and Fermentation of Genetically Engineered Magnetotactic Bacteria.

Biosynthesis provides a critical way to deal with global sustainability issues and has recently drawn increased attention. However, modifying biosynthesized magnetic nanoparticles by extraction is challenging, limiting its applications. Magnetotactic bacteria (MTB) synthesize single-domain magnetite nanocrystals in their organelles, magnetosomes (BMPs), which are excellent biomaterials that can be biologically modified by genetic engineering. Therefore, this study successfully constructed in vivo biotinylated BMPs in the MTB Magnetospirillum gryphiswaldense by fusing biotin carboxyl carrier protein (BCCP) with membrane protein MamF of BMPs. The engineered strain (MSR−∆F−BF) grew well and synthesized small-sized (20 ± 4.5 nm) BMPs and were cultured in a 42 L fermenter; the yield (dry weight) of cells and BMPs reached 8.14 g/L and 134.44 mg/L, respectively, approximately three-fold more than previously reported engineered strains and BMPs. The genetically engineered BMPs (BMP−∆F−BF) were successfully linked with streptavidin or streptavidin-labelled horseradish peroxidase and displayed better storage stability compared with chemically constructed biotinylated BMPs. This study systematically demonstrated the biosynthesis of engineered magnetic nanoparticles, including its construction, characterization, and production and detection based on MTB. Our findings provide insights into biomanufacturing multiple functional magnetic nanomaterials.

Sensitive Detection of Staphylococcus aureus by a Colorimetric Biosensor Based on Magnetic Separation and Rolling Circle Amplification.

Staphylococcus aureus (S. aureus) is a common foodborne pathogen that causes fever, vomiting, and other intestinal symptoms, and seriously affects human health and social safety. As a result, a reliable and sensitive detection technique for S. aureus must be developed. In this work, we proposed a sandwich assay on vancomycin functionalized magnetic beads (Van-MNPs) for S. aureus detection based on the specific binding between IgG and targets. The Van-MNPs were used as a tool for the separation of target bacteria. The biotin-modified IgG mediates binding between DNA nanoflowers (DNFs) and the target bacteria via interacting with streptavidin. The DNFs prepared by rolling circle amplification (RCA) were employed as a nano-container to enhance the capacity of biotins, and the streptavidin-horseradish peroxidase (SA-HRP) was loaded onto DNFs to catalyze the color change of TMB. Therefore, a colorimetric biosensor based on magnetic separation and rolling circle amplification was developed. The proposed methods for S. aureus detection showed a limit of detection (LOD) of 3.3 × 103 CFU/mL and excellent specificity. The biosensor has a certain reference value for the detection of S. aureus in juice.

Immune Cells Activating Biotin-Decorated PLGA Protein Carrier.

Nanoparticle formulations have long been proposed as subunit vaccine carriers owing to their ability to entrap proteins and codeliver adjuvants. Poly(lactic-co-glycolic acid) (PLGA) remains one of the most studied polymers for controlled release and nanoparticle drug delivery, and numerous studies exist proposing PLGA particles as subunit vaccine carriers. In this work we report using PLGA nanoparticles modified with biotin (bNPs) to deliver proteins via adsorption and stimulate professional antigen-presenting cells (APCs). We present evidence showing bNPs are capable of retaining proteins through the biotin-avidin interaction. Surface accessible biotin bound both biotinylated catalase (bCAT) through avidin and streptavidin horseradish peroxidase (HRP). Analysis of the HRP found that activity on the bNPs was preserved once captured on the surface of bNP. Further, bNPs were found to have self-adjuvant properties, evidenced by bNP induced IL-1β, IL-18, and IL-12 production in vitro in APCs, thereby licensing the cells to generate Th1-type helper T cell responses. Cytokine production was reduced in avidin precoated bNPs (but not with other proteins), suggesting that the proinflammatory response is due in part to exposed biotin on the surface of bNPs. bNPs injected subcutaneously were localized to draining lymph nodes detectable after 28 days and were internalized by bronchoalveolar lavage dendritic cells and macrophages in mice in a dose-dependent manner when delivered intranasally. Taken together, these data provide evidence that bNPs should be explored further as potential adjuvanting carriers for subunit vaccines.

Electrochemical genosensor for the specific detection of SARS-CoV-2

Infection caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the Coronavirus disease (COVID-19) and the current pandemic. Its mortality rate increases, demonstrating the imperative need for acute and rapid diagnostic tools as an alternative to current serological tests and molecular techniques. Features of electrochemical genosensor devices make them amenable for fast and accurate testing closer to the patient. This work reports on a specific electrochemical genosensor for SARS-CoV-2 detection and discrimination against homologous respiratory viruses. The electrochemical biosensor was assembled by immobilizing thiolated capture probes on top of maleimide-coated magnetic particles, followed by specific target hybridization between the capture and biotinylated signaling probes in a sandwich-type manner. The probes were rigorously designed bioinformatically and tested in vitro. Enzymatic complexes based on streptavidin-horseradish peroxidase linked the biotinylated signaling probe to render the biosensor electrochemical response. The genosensor showed to reach a sensitivity of 174.4 μA fM−1 and a limit of detection of 807 fM when using streptavidin poly-HRP20 enzymatic complex, detected SARS-CoV-2 specifically and discriminated it against homologous viruses in spiked samples and samples from SARS-CoV-2 cell cultures, a step forward to detect SARS-CoV-2 closer to the patient as a promising way for diagnosis and surveillance of COVID-19.

SARS-CoV-2 electrochemical immunosensor based on the spike-ACE2 complex

Rapid, straightforward, and massive diagnosis of coronavirus disease 2019 (COVID-19) is one of the more important measures to mitigate the current pandemics. This work reports on an immunosensor to rapidly detect the spike protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The immunosensing device entraps the spike protein linked to angiotensin-converting enzyme host receptor (ACE2) protein in a sandwich between carboxylated magnetic beads functionalized with an anti-spike antibody and an anti-ACE2 antibody, further labeled with streptavidin (poly)horseradish peroxidase (HRP) reporter enzyme. The particles were confined at the surface of screen-printed gold electrodes, whose signal resulting from the interaction of the enzyme with a mediator was recorded in a portable potentiostat. The immunosensor showed a sensitivity of 0.83 μA∗mL/μg and a limit of detection of 22.5 ng/mL of spike protein, with high reproducibility. As a proof-of-concept, it detected commercial spike protein-supplemented buffer solutions, pseudovirions, isolated viral particles and ten nasopharyngeal swab samples from infected patients compared to samples from three healthy individuals paving the way to detect the virus closer to the patient.

Interleukin-29 profiles in COVID-19 patients: Survival is associated with IL-29 levels.

Interleukin-29 profiles in COVID-19 patients: Survival is associated with IL-29 levels.

Production of a Natural Dihydropteroate Synthase and Development of a Signal-Amplified Pseudo-Immunoassay for the Determination of Sulfonamides in Pork.

In this study, a type of magnetic photoaffinity-labeled activity-based protein profiling probe for sulfonamide drugs was first synthesized for the purpose of capturing the natural dihydropteroate synthase of Escherichia coli by using simple incubation and magnetic separation. After characterization of its identity with LC-ESI-MS/MS, this enzyme was used as a recognition reagent to develop a direct competitive pseudo-ELISA for the determination of the residues of 40 sulfonamides in pork. Because of the use of streptavidin-horseradish peroxidase and biotinylated horseradish peroxidase as a signal-amplified system, the limits of detection for the 40 drugs were in the range of 0.001-0.016 ng/mL. Compared to the steps in a conventional assay formation, the operation steps were the same, but the sensitivities increased 32-88-fold. Furthermore, the assay performances were better than the previously reported immunoassays performances for sulfonamides. Therefore, this method could be used as a practical tool for multiscreening the trace levels of sulfonamides residues in food samples.

Highly-sensitive and simple fluorescent aptasensor for 17 b-estradiol detection coupled with HCR-HRP structure

As an important kind of environmental endocrine disruptors, 17 β -Estradiol (E2) plays a major role in affecting the growth of human including sexual characters, pregnancy system, etc. In the modern society, with the threat of abuse in breeding, it is imperative to design sensitive methods for detecting low concentration of E2 in environment. In this work, we constructed a highly sensitive and simple fluorescent aptasenor for detecting E2 via amplification of hybridization chain reaction (HCR) and horseradish peroxidase (HRP). Through the competitions between complementary strand (cmDNA) and E2 to E2 aptamer modified on magnetic beads, the unbound cmDNA would be collected and captured by polystyrene microspheres to induce HCR which brought abundant biotin sites. Subsequently, benefit from the excellent catalytic performance of streptavidin-horseradish peroxidase (SA-HRP), the highly sensitive fluorescence signals could be obtained in low concentration of E2. Under the optimal conditions, the prospered method for E2 detection was shown a good liner range from 1 to 100 pg/mL, with the lower detecting limit of 0.2 pg/mL compared with previous work. In addition, the recovery rates tested in the real samples of milk and water were 99.20%–108.06% and 91.07%–106.13%. In all, the assay may provide a perspective way for highly sensitive detection for various contaminants in the real samples.

Highly Selective Electrochemical Detection of 5-Formyluracil Relying on (2-Benzimidazolyl) Acetonitrile Labeling.

The identification of formylpyrimidines in DNA is crucial for a better understanding of epigenetics. Although many techniques have been explored to detect their content, more accurate methods of formylpyrimidine determination are still required due to the relatively lower sensitivity or lack of selectivity in current methods. Herein, an electrochemical method based on the covalent bonding of the azido derivative of (2-benzimidazolyl) acetonitrile (azi-BIAN) and the aldehyde group of 5-formyluracil (5fU) was proposed for the selective detection of 5fU in the presence of 5-formylcytosine (5fC) and apyrimidinic (AP) sites. Target DNA containing 5fU was first treated with azi-BIAN and then incubated with DBCO-PEG4-Biotin to introduce a biotin group by copper-free click chemistry. Next, the sulfhydryl group was attached to the 5' end of above DNA through T4 polynucleotide kinase-catalyzed reaction. Subsequently, the labeled DNA was assembled onto the AuNPs-modified glassy carbon electrode (AuNPs/GCE) through Au-S bonds, and the streptavidin-horseradish peroxidase conjugate (SA-HRP) was further immobilized onto the surface of the above electrode by specific recognition between biotin and streptavidin. Finally, HRP catalyzed hydroquinone oxidation to benzoquinone to enhance the current signal, which was related to the amount of 5fU in nucleic acids. This method demonstrated a good linear relationship with 5fU concentrations ranging from 0.1 to 10 nM. Moreover, the level of 5fU in γ-irradiated nucleic acids was also successfully detected, indicating that the combination of molecule-depended chemical recognition and electrochemical sensing is a promising method for the selective and sensitive detection of 5fU.

Highly sensitive magnetic relaxation sensing method for aflatoxin B1 detection based on Au NP-assisted triple self-assembly cascade signal amplification

Highly sensitive detection of aflatoxin B1 (AFB1) is of great significance because of its high toxicity and carcinogenesis. We propose a magnetic relaxation sensing method based on gold nanoparticles (Au NPs)-assisted triple self-assembly cascade signal amplification for highly sensitive detection of AFB1. Both AFB1 antibody and initiator DNA (iDNA) are labeled on Au NPs to form Ab-Au-iDNA probe. iDNA is enriched by Au NPs to achieve first signal amplification. Different amounts of Ab-Au-iDNA were bound with AFB1 antigen by indirect competitive immunoassay, and then hybridization chain reaction event was initiated by iDNA to produce long hybridization chain reaction products to enrich more horseradish peroxidase-streptavidin for the second signal amplification. Dopamine could be rapidly converted to polydopamine by HRP catalysis, which is used as the third signal amplification. The Fe3+ solution, providing paramagnetic ions with a strong magnetic signal, could be adsorbed by the polydopamine due to the formation of coordination bonds of phenolic hydroxyl groups with Fe3+. This effective interaction between polydopamine and Fe3+ significantly changes the transverse relaxation time signal of Fe3+ supernatant solution, which can be used as a magnetic probe for highly sensitive detection of AFB1. The sensor exhibited high specificity and sensitivity with a detection limit of 0.453 pg/mL owing to the Au NP-assisted triple self-assembly cascade signal amplification strategy. It has been successfully employed for AFB1 detection in animal feed samples with consistent results of enzyme linked immune sorbent assay and high-performance liquid chromatography.

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout.

To date, medical diagnosis of gout and pseudogout has been performed by observing the crystals in the joint fluid of patients under a polarized microscope. Conventional diagnostic methods using a polarized microscope have disadvantages, such as time-consuming analysis, a high false negative rate, and difficulty in distinguishing gout with monosodium urate (MSU) crystals and pseudogout with calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluids. In this study, a chromogenic assay for the diagnosis of gout and pseudogout, without the requirement of a polarized microscope and trained experts, was proposed using Fv antibodies with specific binding activities to MSU and CPPD crystals. The IgG VH chain Fv library with randomized complementarity-determining region 3 (CDR3) region was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv antibodies with binding activity to MSU and CPPD crystals were screened from the autodisplayed Fv library on the E. coli outer membrane, and five clones were selected. On the basis of the binding properties of the screened Fv antibodies, peptides with the selected clone of amino acid sequences of the CDR3 region (15 residues) were chemically synthesized. The binding properties of the synthetic peptides with amino acid sequences of CDR3 regions from the selected clones were analyzed using fluorescence imaging and flow cytometry, and the affinity constants (Kd) of each peptide for binding to MSU and CPPD crystals were calculated by fitting based on the isotherm model. A chromogenic assay configuration for gout and pseudogout was developed using synthetic peptides. In this chromogenic assay, synthetic peptides labeled with biotin and streptavidin-horseradish peroxidase (HRP) complex were used, and crystal detection was possible using a chromogenic reaction between HRP and a chromogenic substrate (TMB). Finally, gout and pseudogout were diagnosed by detecting MSU and CPPD crystals in the synovial fluid in the concentration range of 0-300 μg/mL.

Development and Optimization of a Rapid Colorimetric Membrane Immunoassay for Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 103-105 bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without crossreactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Dual-Mode Aptasensor Assembled by a WO3/Fe2O3 Heterojunction for Paper-Based Colorimetric Prediction/Photoelectrochemical Multicomponent Analysis.

The programed bimodal photoelectrochemical (PEC)-sensing platform based on DNA structural switching induced by targets binding to aptamers was innovatively designed for the simultaneous detection of mucin 1 (MUC1) and microRNA 21 (miRNA-21). To promote excellent current intensity as well as enhance the sensitivity of aptasensors, the evenly distributed WO3/Fe2O3 heterojunction was prepared as a transducer material, notably reducing the background signal response and extending the absorption of light. The multifunctional paper-based biocathode was assembled to provide a visual colorimetric assay. When introducing the integrated signal probe (ISP) composed of signal probe 1 (sP1) and signal probe 2 (sP2) on paper-based working units modified with gold nanoparticles (AuNPs), recognition sites of two targets were formed. In the presence of MUC1 protein, both sP1 and the target on the working unit were released into the corresponding colorimetric unit because of the DNA specific recognition. The horseradish peroxidase-streptavidin (HRP-SA) carried by free sP1 could oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to turn a blue-colored oxidized TMB (oxTMB) in the presence of hydrogen peroxide (H2O2), which ultimately gained a higher photocurrent signal. Furthermore, miRNA-21 was modified on another working unit by binding with sP2, leading to changes in the current signal and thus enabling real-time detection of analytes with the assistance of a digital multimeter. The PEC aptasensor offered a wide dynamic range of 10 fg·mL-1-100 ng mL-1 for MUC1 and 0.1 pM-10 nM for miRNA-21, with a low detection limit of 3.4 fg·mL-1 and 36 fM, respectively. It laid the foundation for synchronous detection of multiple analytes and initiated a new way for the enhancement in modern next-generation disease diagnosis.

Electrochemical immunoplatform to assist in the diagnosis and classification of breast cancer through the determination of matrix-metalloproteinase-9

Matrix metalloproteinase 9 (MMP-9) is a zinc-dependent endopeptidase that promotes angiogenesis, tumor growth, metastasis and cell invasion through the degradation of extracellular matrix. This work reports a magnetic microbeads (MBs)-based sandwich immunoassay for the amperometric determination of MMP-9 at screen-printed carbon electrodes (SPCEs). The suitable capture antibody (cAb) is immobilized onto carboxylic MBs to selectively capture the antigen which is sandwiched with a biotinylated detector antibody (biotin-dAb) further conjugated with a commercial streptavidin-horseradish peroxidase (Strep-HRP) polymer. This immunoplatform provides great analytical characteristics in terms of selectivity and sensitivity, achieving a LOD value of 2.4 pg mL−1 for standards in buffered solutions. Although this value is similar to those reported for some other approaches described so far, the method described here is simpler involving a single 30 min incubation step which makes it ideal for automation or implementation in POC devices. Moreover, the method was assayed for the accurate determination of endogenous MMP-9 in both cancer cell lysates and serum samples of patients diagnosed with different subtypes of breast cancer (BC) after a simple dilution. The results obtained show that the disposable and affordable immunoplatform developed is able not only to discriminate BC patients from healthy individuals but also to do it for the worst outcome triple negative (TNBC) subtype.