Simultaneous Detection Of Multiple Targets Research Articles

Advancements and applications of loop-mediated isothermal amplification technology: a comprehensive overview.

Loop-mediated isothermal amplification (LAMP) is a novel method for nucleic acid detection known for its isothermal properties, high efficiency, sensitivity, and specificity. LAMP employs 4 to 6 primers targeting 6 to 8 regions of the desired sequence, allowing for amplification at temperatures between 60 and 65°C and the production of up to 109 copies within a single hour. The product can be monitored by various methods such as turbidimetry, fluorometry, and colorimetry. However, it faces limitations such as the risk of non-specific amplification, challenges in primer design, unsuitability for short gene sequences, and difficulty in multiplexing. Recent advancements in polymerase and primer design have enhanced the speed and convenience of the LAMP reaction. Additionally, integrating LAMP with technologies like rolling circle amplification (RCA), recombinase polymerase amplification (RPA), and CRISPR-Cas systems has enhanced its efficiency. The combination of LAMP with various biosensors has enabled real-time analysis, broadening its application in point-of-care testing (POCT). Microfluidic technology has further facilitated the automation and miniaturization of LAMP assays, allowing for the simultaneous detection of multiple targets and preventing contamination. This review highlights advancements in LAMP, focusing on primer design, polymerase engineering, and its integration with other technologies. Continuous improvements and integration of LAMP with complementary technologies have significantly enhanced its diagnostic capabilities, making it a robust tool for rapid, sensitive, and specific nucleic acid detection with promising implications for healthcare, agriculture, and environmental monitoring.

Simultaneous multiple target detection platform based on vertical flow immunoassay

In general, vertical flow assay (VFA) has a disadvantage of requiring a complex analysis process that involves manually injecting various reagents (target analyte, washing buffer, detection conjugate, etc.) sequentially. However, in this study, we have developed an innovative paper-based VFA device that replaces the complex analysis process with one-step and enables the detection of multiple targets. The fabrication process of the multi-target detection VFA device is as follows: preparation and pre-treatment of the strip materials, design of strip cartridge, design of the multiple detection VFA device, optimization experiments for strip sample flow rates, determination of device analysis time, determination of device limit of detection (LOD), multiple target signal uniformity experiment, immunoglobulin G (IgG) and C-reactive protein (CRP) antigen-antibody multiple detection experiment, and data extraction and analysis method. The use of paper-based materials enables the device to be produced at cost-effective, and cartridge production allowed for uniform array formation. IgG and CRP are used to evaluate the performance of the device as common biomarkers. The device proposed in this study is currently under research. To validate multiple target detection capability of the VFA device proposed in this study, two types of antigens-antibodies (Human IgG and Human CRP) were employed. The detection limit is 0.15 μg/mL for IgG and 0.19 μg/mL for CRP in naked eye. Furthermore, it was confirmed that there is no cross-reactivity caused by the device structure through IgG and CRP antigens. In conclusion, the VFA device proposed in this study consists of a one-step analysis process, and it has been confirmed that it can detect multiple targets simultaneously.

Color- and background-free Raman-encoded lateral flow immunoassay for simultaneous detection of carbendazim and imidacloprid in a single test line

Multiple detection based on lateral flow immunoassay (LFA) holds significant research importance across various fields. Recently, the use of multi-color surface-enhanced Raman scattering (SERS) nanotags has enabled simultaneous detection of multiple targets within a single test line (T-line) in LFA, greatly enhancing detection efficiency, reliability, and application flexibility. However, challenges regarding signal overlap in the Raman-fingerprint region (400–1800 cm−1) and the difficulty in determining specific targets based on typical color analysis have persisted. In this study, we developed two types of nanoprobes that encode both color and Raman information to address these challenges in simultaneous multiple detection on a single T-line in SERS-LFA. The nanoprobes were encoded with distinguishable blue and red colors, respectively, enabling colorimetric study in multiplex LFA for qualitative analysis. Additionally, they were encoded with distinctive and non-overlapping Raman signals in the Raman-silent region (1800–2800 cm−1), ensuring accurate and sensitive detection by effectively avoiding interference from each other as well as background signals in the sample matrix. Using carbendazim (CBZ) and imidacloprid (IMI) as model targets, the color- and Raman-encoded SERS-LFA demonstrated promising potential for the simultaneous detection of CBZ and IMI in both standard and real samples, whether in a colorimetric or a SERS mode.

Shared hairpin structure electrochemiluminescence biosensor based on Au@Ni-Co metal organic frameworks for simultaneous detection of Pb(II) and S.aureus

The excessive content of lead (Pb(II)) and Staphylococcus aureus (S.aureus) seriously harms the quality of aquatic products. In this paper, a highly sensitive electrochemiluminescence (ECL) biosensor was constructed using the synergistic effect of Au NPs@Nickel-Cobalt-Metal-organic frameworks (Au@Ni-Co-MOFs) and double potential resolution function of urchin-like Au@luminol and Cadmium sulfide quantum dots (CdS QDs) for synchronous detection of Pb(II) and S.aureus in aquatic products. Au@Ni-Co-MOFs as the base material, its cube structure can improve the surface active area and sensitivity of the sensor, providing more catalytic active sites for the two functional probes. Urchin-like Au@luminol binding aptamer DNA2 specifically recognizes Pb(II), CdS QDs binding aptamer DNA3 specifically recognizes S.aureus, which collaboratively catalyzed hydrogen peroxide reduction to produce two electrochemiluminescence signals. The shared hairpin structure DNA1 binds stably to Au@Ni-Co-MOFs via the Au-S bond, and the two functional probes are complementary paired with the DNA1 respectively to ensure the specificity of the aptamer. According to the ECL intensity changes of different potentials signal sources, the synchronous detection of Pb(II) and S.aureus with different concentrations is realized. The sensor realizes the detection of two targets in aquatic products and provides a new strategy for the simultaneous detection of multiple targets.

A novel surface-enhanced Raman based molecular identification platform for multiplexed and highly accurate clinical diagnosis of viral diseases.

During the infection process, the interactions among respiratory viruses impact the dynamics of transmission and clinical outcomes. Therefore, efficient molecular detection methods provide a basis for rational drug use and effective health management. Surface-enhanced Raman scattering (SERS) is an ultra-sensitive spectroscopic technique capable of generating extremely narrow spectra (∼1-2 cm-1), enabling simultaneous detection of multiple targets. By judiciously designing plasmonic nanostructures as SERS substrates, Raman signals can be amplified by several orders of magnitude (∼105-1015), facilitating the detection of trace biomolecules. In this highlight, we highlight the work about a novel SERS platform for the high-precision multi-virus molecular identification. This may offer a highly sensitive, specific, and accurate method for the detection of multiple viruses.

Cas12a-based direct visualization of nanoparticle-stabilized fluorescence signal for multiplex detection of DNA methylation biomarkers

The CRISPR-Cas12a RNA-guided complexes hold immense promise for nucleic acid detection. However, limitations arise from their specificity in detecting off-targets and the stability of the signal molecules. Here, we have developed a platform that integrates multiplex amplification and nanomolecular-reporting signals, allowing us to detect various clinically relevant nucleic acid targets with enhanced stability, sensitivity, and visual interpretation. Through the electrostatic co-assembly of the Oligo reporter with oppositely charged nanoparticles, we observed a significant enhancement in its stability in low-pollution environments, reaching up to a threefold increase compared to the original version. Additionally, the fluorescence efficiency was expanded by three orders of magnitude, broadening the detection range considerably. Utilizing a multiplex strategy, this assay can accomplish simultaneous detection of multiple targets and single-point indication detection of nine specific targets. This significant advancement heightened the sensitivity of disease screening and improved the accuracy of diagnosing disease-related changes. We tested this assay in a colorectal cancer model, demonstrating that it can identify DNA methylation features at the aM-level within 40–60 min. Validation using clinical samples yielded consistent results with qPCR and bisulfite sequencing, affirming the assay's reliability and potential for clinical applications.

Highly Sensitive Naked Eye Detectable Colorimetric Biosensors Made from Macroporous Framework Melamine Foams for Onsite and Simultaneous Detection of Multiple Environmental Hazards in Flowing Through Sensing Systems

AbstractPersonal‐use, naked eye‐readable, low‐cost, highly sensitive, and selective biosensors for rapid detection of environmental toxicants are relevant for many application scenarios. Here, the recent developments of highly sensitive and naked eye distinguishable colorimetric sensors by using commercially available melamine foam (MF) as basic sensing materials for instant and volume‐responsive simultaneous detection of multiple targets in fluid systems are reported. The MF possesses a unique reticulated three‐dimensional (3D) macroporous framework structure enabling rapid mass transfer of large biomolecules through the structures in all directions, ensuring easy access of numerous active binding sites of the chemically modified framework to the proteins and target molecules, and subsequently providing significantly increased sensitive and volume‐responsive detection of target molecules in flowing through sensor systems. Promising results in direct, sandwich, and competitive ELISA tests demonstrated the great application potential of the materials. Besides, an additive and simultaneous detection of two targets in one system is achieved by using different layers of the sensor materials in a flowing‐through filtering device. The novel biosensors are expected to significantly improve the sensitivity and broaden the applications of ELISA in the rapid detections of trace amounts of toxicants in liquid and aerosol systems.

Multi-target detection and sizing of single nanoparticles using an optical star polygon microcavity.

We present a miniaturized single nanoparticle detector that utilizes an optical star polygon microcavity with a 3 µm-radius. The microcavity supports high-quality factor resonant modes, with light localized at the corners of the star-shaped polygon, where the air region is situated. When nanoparticles are positioned at the corners of the microcavity, the light-matter interactions are enhanced. Notably, increasing the number of particles has little effect on the quality factor of the cavity, making it ideal for the simultaneous detection of multiple targets. Our numerical simulations demonstrate the high precision detection of polystyrene nanoparticles with a radius of 3 nm using this method. Furthermore, the size and number of nanoparticles can be determined by utilizing the triangular corners of the cavity as rulers. These findings represent a significant advancement in miniaturized and multi-target simultaneous nanoparticle detection. The proposed detector is expected to have a wide range of applications in various fields, including biomedicine and environmental monitoring.

Simultaneous Detection of Staphylococcus aureus and E. coli O157:H7 Using a Self-Calibrated Potentiometric Sensors Array Based on Peptide Recognition

A novel self-calibrated potentiometric sensors array for simultaneous detection of staphylococcus aureus (SA) and E. coli O157:H7 (E. coli) based on peptide recognition have been proposed on a 4-channel screen printed carbon electrode (SPCE) modified with DenAu and PTAA. Two specific peptides for recognition of SA and E. coli were assembled on two working channels, respectively. For two calibration channels, two non-specific peptides with the same isoelectric point and length as the specific ones were coated on the surface to eliminate the influence of the background matrix on the potentiometric responses of working channels, and improve the detection accuracy. Under the optimal conditions, the sensors array exhibited high sensitivity to SA and E. coli in the range of 1.0×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> CFU/mL - 1.0×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">6</sup> CFU/mL and 1.0×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> CFU/mL - 1.0×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">5</sup> CFU/mL with the detection limits of 38 CFU/mL and 241 CFU/mL, respectively, and also good specificity and repeatability. Furthermore, it was successfully applied to the detection of milk samples, providing an effective strategy the on-site simultaneous detection of multiple targets.

Detection and quantification of integrated vector copy number by multiplex droplet digital PCR in dual-transduced CAR T cells

The success of chimeric antigen receptor (CAR) Tcell therapies in refractory hematologic malignancies has prompted investigation of their efficacy in solid tumors. AUTO6NG is a dual-transduced GD2-targeting CAR that encodes distinct modules designed to enhance Tcell activity in relapsed/refractory neuroblastoma. The ability to detect and precisely quantify vector copy number (VCN) for each integrated vector is essential for assessing the effect of each module on Tcell tumor infiltration, persistence, and clinical activity. Droplet digital PCR (ddPCR) enables accurate, sensitive, and absolute quantification of specific nucleic acid sequences. Compared to standard detection of two targets, multiplex ddPCR assays allow simultaneous detection of up to four targets by selective modulation of signal amplitude while retaining the ability to quantify the target. We have developed a multiplex assay based on the two-channel system for simultaneous detection and quantification of three targets in AUTO6NG CAR Tcells. The assay was highly specific, sensitive, accurate, and reproducible across time and samples. No differences were observed in measuring VCN between standard duplex and multiplex assays. Our results demonstrate that ddPCR is an accurate and cost-effective method for simultaneous detection of multiple targets in genomic DNA derived from engineered CAR Tcells.

Deep Domain Adaptation Enhances Amplification Curve Analysis for Single-Channel Multiplexing in Real-Time PCR.

Data-driven approaches for molecular diagnostics are emerging as an alternative to perform an accurate and inexpensive multi-pathogen detection. A novel technique called Amplification Curve Analysis (ACA) has been recently developed by coupling machine learning and real-time Polymerase Chain Reaction (qPCR) to enable the simultaneous detection of multiple targets in a single reaction well. However, target classification purely relying on the amplification curve shapes currently faces several challenges, such as distribution discrepancies between different data sources of synthetic DNA and clinical samples (i.e., training vs testing). Optimisation of computational models is required to achieve higher performance of ACA classification in multiplex qPCR through the reduction of those discrepancies. Here, we proposed a novel transformer-based conditional domain adversarial network (T-CDAN) to eliminate data distribution differences between the source domain (synthetic DNA data) and the target domain (clinical isolate data). The labelled training data from the source domain and unlabelled testing data from the target domain are fed into the T-CDAN, which learns both domains' information simultaneously. After mapping the inputs into a domain-irrelevant space, T-CDAN removes the feature distribution differences and provides a clearer decision boundary for the classifier, resulting in a more accurate pathogen identification. Evaluation of 198 clinical isolates containing three types of carbapenem-resistant genes (blaNDM, blaIMP and blaOXA-48) illustrates a curve-level accuracy of 93.1% and a sample-level accuracy of 97.0% using T-CDAN, showing an accuracy improvement of 20.9% and 4.9% respectively, compared with previous methods. This research emphasises the importance of deep domain adaptation to enable high-level multiplexing in a single qPCR reaction, providing a solid approach to extend qPCR instruments' capabilities without hardware modification in real-world clinical applications.

A Multiplexed SERS Microassay for Accurate Detection of SARS-CoV-2 and Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold-silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/μL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care

Loop-mediated isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-time fluorescence detection using fluorophore-labelled probes attached to oligonucleotide sequences complementary to the target nucleic acid. This methodology allows the simultaneous detection of multiple targets (multiplexing) in one LAMP assay. A method for multiplexing LAMP is the amplification by release of quenching (DARQ) technique by using a 5'-quencher modified LAMP primer annealed to 3'-fluorophore-labelled acting as detection oligonucleotide. The main application of multiplex LAMP is the rapid and accurate diagnosis of infectious diseases, allowing differentiation of co-infecting pathogens in a single reaction. Schistosomiasis, caused among other species by Schistosoma mansoni and strongyloidiasis, caused by Strongyloides stercoralis, are the most common helminth-parasite infections worldwide with overlapping distribution areas and high possibility of coinfections in the human population. It would be of great interest to develop a duplex LAMP to detect both pathogens in the same reaction. In this study, we investigate the use of our two previously developed and well-stablished LAMP assays for S. mansoni and Strongyloides spp. DNA detection in a new duplex real-time eight-primer system based on a modified DARQ probe method that can be performed in a portable isothermal fluorimeter with minimal laboratory resources. We also applied a strategy to stabilize the duplexed DARQ-LAMP mixtures at room temperature for use as ready-to-use formats facilitating analysis in field settings as point-of-care diagnostics for schistosomiasis and strongyloidiasis.

Target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a for simultaneous detection of HPV16 and HPV18.

Simultaneous detection of multiple targets can provide important data support for clinical diagnosis and treatment. Here, we report a facile isothermal assay based on target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a (TM-RCT/Cas12a-Cas13a). Through facile one-step amplification (TM-RCT), two target DNAs are converted to RNA amplified products. The simultaneous detection of HPV16 and HPV18 is then achieved by combining two CRISPR/Cas systems. This system shows excellent sensing performance and provides a universal method for simultaneous detection.

Freeze-synthesized bio-barcode immunoprobe based multiplex fluorescence immunosensor for simultaneous determination of four nitrofuran metabolites

Here we have reported a simple and sensitive bio-barcode immunosensor for simultaneous detection of 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), and semicarbazide (SEM) in aquatic products. According to freeze–thaw strategy, four fluorophores (FAM, HEX, ROX, Cy5) labeled single-stranded DNA (ssDNA) were conjugated onto the surface of gold nanoparticles (AuNPs) with corresponding four nitrofuran metabolites monoclonal antibodies (mAbs) for forming four bio-barcode fluorescence immunoprobes. The fluorescence of immunoprobes was quenched by AuNPs. In test progress, the ssDNA with fluorophores were released by adding the dithiothreitol (DTT) and the fluorescence recovered. The immunosensor exhibited sensitive and specific detection of nitrofuran metabolites from 0.05 to 28 μg/L. The limit of detection (LOD) was 0.01, 0.02, 0.02, and 0.05 μg/L for AOZ, AMOZ, AHD, and SEM, respectively. The recoveries of four nitrofuran metabolites in spiked aquatic products have been confirmed by UPLC-MS/MS. The bio-barcode based multiplex immunosensor provides a promising strategy for simultaneous detection of multiple targets.

A Multiple-Target Simultaneous Detection Method for Immunosorbent Assay and Immunospot Assay.

Enzyme-linked immunosorbent assay (ELISA) is one of the most common methods in biological studies, and enzyme-linked immunospot (ELISpot) is a method to measure specific cell numbers by detecting protein secretion at a single-cell level. However, these two current methods can only detect one signal at one time and the sensitivity is not high enough to test low-concentration samples, which are major shortcomings in systematically analyzing the samples of interest. Herein, we demonstrated fluorescence-based oligo-linked immunosorbent assay (FOLISA) and fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification. Signal amplification and simultaneous multiple-target detection were achieved by DNA complementary pairing and modular orthogonal DNA concatemers. By comparing FOLISA with traditional ELISA and comparing FOLISPOT with traditional ELISPOT, we found that the detection sensitivities of FOLISA and FOLISPOT are much higher than those of traditional ELISA and ELISPOT. The detection limit of ELISA is around 3 pg/mL, and the detection limit of FOLISA is below 0.06 pg/mL. FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable for ELISPOT. Furthermore, FOLISA and FOLISPOT allowed sequential detection of multiple targets by using a single dye or multiple dyes in one round and sequential detection in multiple rounds. Thus, FOLISA and FOLISPOT enabled simultaneous detection of a large number of targets, significantly improved the detection sensitivity, and overcame the shortcomings of ELISA and ELISPOT. Overall, FOLISA and FOLISPOT presented effective and general platforms for rapid and multiplexed detection of antigens or antibodies with high sensitivity, either in laboratory tests or potentially in clinic tests.

Intelligently identifiable membrane immunochip sensor based on Braille-like code for simultaneous multi-veterinary drug detection

There may be risk of multiple veterinary drug residues in one food sample; thus, a key challenge for the simultaneous detection of multiple targets was raised in food safety analysis. In this study, a multi-dot membrane immunochip sensor (MIS) based on Braille-like code was proposed for the sensitive, specific, and simultaneous detection of four veterinary drugs: sulfamethazine (SMZ), amantadine (AMD), danfloxacin (DAN), and tylosin (TYL). The 16 types of residual situations for four veterinary drugs can be qualitatively and quantitatively analyzed through the Braille-like code and signal intensity of dots. In addition, an Android application was exploited to intelligently identify the Braille-like code pattern; thus, the residue situations of the corresponding veterinary drugs can be automatically output by a smartphone. The cutoff values were 25 ng/mL for the four veterinary drugs by the naked eye. The limits of detection for SMZ, AMD, DAN, and TYL were calculated as 0.22, 0.058, 0.29, and 0.14 ng/mL, respectively. To verify the performance of this multi-dot MIS, spiked chicken samples with different concentrations were tested, and the results verified the accuracy and precision of this method. The average spiked recoveries were 79.2–116.7%, and the coefficient of variation was 1.6–10.5%. Flexible, versatile and intelligent discriminant methods give this multi-dot MIS great potential to rapidly recognize complex situations in the field of simultaneous detection of analytes.

A facile “turn-on” fluorescent aptasensor for simultaneous detection of dual mycotoxins in traditional Chinese medicine based on graphene oxide and FRET

Mycotoxin is a common sort of harmful contaminant in traditional Chinese medicine (TCM), which is in a great demand of controlling. On this account, a facile “turn-on” fluorescent aptasensor based on fluorescence resonance energy transfer (FRET) for simultaneous detection of patulin (PAT) and zearalenone (ZEN) was developed. In this study, the aptamers of PAT and ZEN were labeled by FAM and Cy3, respectively, serving as fluorescence probes. Both aptamers could adsorb on the surface of graphene oxide (GO) via π-π stacking, which will consequently result in the occurrence of FRET between the fluorophores and GO. In the absence of the targets, the fluorescence would be quenched “off”. In the presence of any of the dual mycotoxins, the corresponding aptamers would interact with the targets and release from GO due to the conformational variation, leading to a fluorescence “turn-on” effect. The limit of detection of this difunctional aptasensor was 2.29 nM for PAT and 0.037 nM for ZEN, respectively. This aptasensing platform exhibited satisfactory selectivity against interferents and reliability in real TCM sample detection. To our knowledge, it is the first aptasensor based on GO and FRET that realizes simultaneous detection of dual mycotoxin in TCM. Moreover, the measurement takes merely ∼60 min, does not need complicated pretreatment, and uses only inexpensive aptamer and GO as consuming materials. To sum up, this aptasensor exhibits great potential in fast, cost-effective and reliable simultaneous detection of multiple targets, and is expected to contribute to the quality and safety control of TCM.

Effect of Non-surgical Periodontal Therapy on Viruses- HSV, EBV and HCMV using Multiplex PCR in Chronic Periodontitis Patients.

Periodontitis, the most common periodontal pathology, is a chronic disease orchestrated amongst the infectious organisms and humoral and cellular immune responses of the host.2 Initially only the bacteria like Treponema Denticola, PorphyromonasGingivalisand Tannerella Forsythia were linked with the disease and hence the term periodontal microbiology was recoined as periodontal bacteriology. The hunt for the etiology however did not end here as many patients still did not fall into this domain of causative factors. This tickled the researchers to put on their thinking caps when they pondered over viruses being the unexplained etiology. Multiplex PCR is the simultaneous detection of multiple targets in a single reaction well, with a different pair of primers for each targets. This technique is indispensable with two or more probes that can be distinguished from each other and recognized simultaneously.

Reusable OIRD Microarray Chips Based on a Bienzyme-Immobilized Polyaniline Nanowire Forest for Multiplexed Detection of Biological Small Molecules.

Quantitative detection of multiple biological small molecules is critical for health evaluation and disease diagnosis. In this study, a microarray chip featuring a bienzyme-immobilized polyaniline nanowire forest on fluorine-doped tin oxide (bienzyme-PANI/FTO) is developed for this purpose. On such a chip, the target molecules are oxidized under the catalysis of their attached oxidases to produce hydrogen peroxide, which further induces the partial oxidation of local PANI nanowires in the presence of horseradish peroxidase (HRP) enzyme. The redox state change of PANI nanowires is monitored by the oblique incident reflectivity difference (OIRD) technique in a real-time and wireless manner, thus allowing for quantitative analysis of the target molecules. As typical model targets, hydrogen peroxide, glucose, lactic acid, and cholesterol are successfully detected with low detection limits, excellent specificities, and broad detection ranges, all of which fully meet the requirements for clinical analysis of human serum samples. Simultaneous detection of multiple targets on an individual chip is further demonstrated using the OIRD scanning mode. Meanwhile, by simple electrochemical reduction of the PANI nanowires, the chip is reusable for more than eight detection cycles without evident decay in its performance. The detection principle of this chip is also universal to other small molecules, and thus, it shows great promise as a valuable device to analyze biological small molecules.