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In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

A real-time centrifugal microfluidic chip with a companion analyzer was developed for highly sensitive, multiplexed nucleic acid detection based on RPA (recombinase polymerase amplification) isothermal amplification. In order to improve the detection sensitivity, two different optimization strategies were systematically studied. Witnessing the high viscosity of RPA reagent, one way was to improve the amplification efficiency by intentionally introducing active mixing based on centrifugal actuation. While the other way was to improve the detection sensitivity by utilizing two-stage amplification. The templates were pre-amplified in the first-stage amplification chamber before they were aliquoted and distributed into a couple of second-stage ones for multiplexed detection. Different mixing methods relative to different actuation time were studied and compared. Similarly, different two-stage amplification modes relative to different time protocols were compared as well. Totally four different amplification modes including with or without mixing, and with or without two-stage amplification, were systematically analyzed and compared. It was found that, the detection sensitivity could be significantly improved by the two-stage amplification with active mixing. Furthermore, as a proof of concept, the performance of the developed microfluidic chip was demonstrated by successfully detecting different genes of African swine fever virus (ASFV) in parallel with high sensitivity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The integration of a microfluidic sample delivery chip with a silicon nanowire (SiNW) biosensor device for multiplexed biomolecule detection was demonstrated. The SiNWs and polydimethylsiloxane (PDMS) chips were fabricated by complementary metal oxide semiconductor (CMOS) compatibility methods at a low cost. They were integrated together by using optimal O2 plasma parameters that enabled rapid and leakage-free bond formation, without additional heating or applied pressure. The capillary action induced by the hydrophilicity of the channels using polyvinylpyrrolidone (PVP) was demonstrated to allow analyte solution delivery onto the sensor array directly, without the need of using external pumping devices. The multiplexed, real-time, and label-free electrical biomolecule detection was demonstrated with high sensitivity and selectivity. The novel detection approach enables simple and rapid multiplexed biomarker detection, showing great promise for application in point-of-care disease diagnostics. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Label-free and enzyme-free multiplexed detection of TFs is first realized. • PPyNPs is first applied in detection of transcription factors. • Absorption and desorption process between DNA-AgNCs and PPyNPs is first described. • Multiplexed detection of NF-κB p50 and p53 in cell nuclear extracts is implementable. • Facile evaluation of TFs inhibitors is realized. Abstract Transcription factors (TFs) regulate information flow from gene to protein, and they are recognized as key indicators to reflect cellular processes. Facile monitoring of TFs may aid diagnostics and treatment, but we still lack of techniques for highly efficient detection of them. In this research, a hybrid material consisting of DNA-silver nanoclusters (DNA-AgNCs) and polypyrrole nanoparticles (PPyNPs) was built for TFs detection. The designed DNA-AgNCs have a hairpin-shaped nucleic acid architecture with a double-stranded stem for recognizing TFs and a single-stranded loop for interacting with PPyNPs. In the absence of TFs, DNA-AgNCs are absorbed to PPyNPs, resulting in fluorescent quenching. While in the presence of TFs, the binding between TFs and the DNA-AgNCs caused desorption of DNA-AgNCs via steric hindrance mechanism. Accordingly, the increase of fluorescence derived from desorption is used for quantifications. Derived from low non-specific protein absorption features of PPyNPs, detection limit of 70 pM for NF-κB p50 and 110 pM for p53 were obtained. Then, by absorbing two kinds of DNA-AgNCs to PPyNPs, label-free multiplexed detection of TFs was first realized. Additionally, we suggested that this platform can be developed for drug screening by evaluating inhibitory effect of a pair of optical isomers towards TFs. [ABSTRACT FROM AUTHOR]

A fluorescent suspension array combining digital encoding and molecular imprinting technologies was applied for multiplexed detection of phenols in aqueous medium. In this assay, nanomaterials such as silver (Ag), cuprous oxide (Cu 2 O), magnesium oxide (MgO), and zinc oxide (ZnO) were assembled on microbeads as digital encoding signal sources, then, (Digital encoding signal sources were produced by assembling silver (Ag), cuprous oxide Cu2O), magnesium oxide (MgO), and zinc oxide (ZnO) on microbeads. Molecular imprinted nanoparticles based on CdSe/ZnS quantum dots with various emission wavelengths (525, 565 and 585 nm) were grafted on the encoded beads through electrostatic adsorption and were used as the sensing units. The decoding spectra of the suspension array were stimulated and collected by a home-built laser induced breakdown spectroscopy system. The resulted spectra were transformed into digital sequences for encoding. As demonstrated in the gradient detections to various phenol solutions in concentration range of 0.98–62.5 μg/mL, the functionality of the suspension array was proven in the control experiments between the parent sensor beads and the molecular imprinting silica nanoparticles assembled sensor beads. Furthermore, the selectivity of suspension array was verified by the mixed adsorption experiments conducted in multicomponent analytes. [ABSTRACT FROM AUTHOR]

Abstract: Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Herein, we show that the combination of a sandwich assay and DNA‐assisted nanopore sensing could unambiguously identify and quantify several antigens in a mixture. We use five barcode DNAs to label different gold nanoparticles that can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer‐associated biomarkers within a very small sample volume, which is critical for non‐invasive early diagnosis of cancer. [ABSTRACT FROM AUTHOR]

This communication is the first to demonstrate the high throughput power of a 30 mm in length reaction flow (RF) column to conduct a targeted analysis of phenolics in a complex sample matrix (tea) utilising post-column derivatisation (PCD). Sensitivity, speed and efficiency could be tuned via the selection of the RF column separation flow rate. RF-PCD compared to conventional PCD approach with a 100 μL loop resulted in a gain of up to 150% in sensitivity. The reaction loop volumes employed in conventional PCD approaches will always be the limitation for compatibility with low dispersion chromatography systems and/or columns. [ABSTRACT FROM AUTHOR]

Mycotoxins threaten human health seriously because they usually exist in food, fodder and commodities. In this study, a rapid and sensitive immunoassay system for commonly encountered mycotoxins was established based on microfluidics and protein microarrays. Four mycotoxins (T-2 toxin, aflatoxin B1, ochratoxin A, and zearalenone) can be automatically detected in a custom-made microdevice within 30 min under the assistance of a prototype of the instrument with a fluid control system and an imaging system. Once the microdevices are fabricated, they are small-sized and user-friendly. Standard curves for each of the studied mycotoxins were generated with a good logistic correlation (R 2 > 0.98). Working ranges from 0.1 to 20 ng/ml were employed in the immunoassay being the limit of detection achieved between 0.03 and 1.24 ng/ml. These values were calculated when the four mycotoxins were present in samples at the same time. Samples of spiked water and field corn were tested to assess the performance of our microfluidic-based detection technique for the mycotoxins. Recovery rates of mycotoxins from spiked water and corn samples were accessed and the results ranged from 80% to 110%, where the intra-assay coefficients of variation were under 15%. In summary, the system can realize rapid and reliable detection of multiple contaminants in actual samples automatically. [ABSTRACT FROM AUTHOR]

This paper reports the multiplexed monitoring of two promising biomarkers, beta-amyloid (Aβ42) and total tau (T-tau), in both buffer and cerebrospinal fluid (CSF) for Alzheimer's disease (AD) using label-free optical nanosensors. It has been found that 7.8 pg/ml of Aβ42 in buffer and 15.6 pg/ml of T-au in buffer can be readily detected with very good specificity. Based on our measurements, the purchased CSF itself contains Aβ42, whose concentration is estimated to be about 400 pg/ml. Aβ42 and T-tau in the mixtures of Aβ42 and T-tau spiked in CSF have been detected successfully, indicating the feasibility of the optical nanosensors to detect these biomarkers in clinical samples. For the measurements, only a small amount (~1 μl) of the samples is required. This type of sensor is suitable for point-of-care application to diagnose the AD due to its low cost and ease-of-operation. [ABSTRACT FROM AUTHOR]

We propose a new analytical scheme in which field-flow fractionation (FFF)-based separation of target-specific polystyrene (PS) particle probes of different sizes are incorporated with amplified surface-enhanced Raman scattering (SERS) tagging for the simultaneous and sensitive detection of multiple microRNAs (miRNAs). For multiplexed detection, PS particles of three different diameters (15, 10, 5 μm) were used for the size-coding, and a probe single stranded DNA (ssDNA) complementary to a target miRNA was conjugated on an intended PS particle. After binding of a target miRNA on PS probe, polyadenylation reaction was executed to generate a long tail composed of adenine (A) serving as a binding site to thymine (T) conjugated Au nanoparticles (T-AuNPs) to increase SERS intensity. The three size-coded PS probes bound with T-AuNPs were then separated in a FFF channel. With the observation of extinction-based fractograms, separation of three size-coded PS probes was clearly confirmed, thereby enabling of measuring three miRNAs simultaneously. Raman intensities of FFF fractions collected at the peak maximum of 15, 10 and 5 μm PS probes varied fairy quantitatively with the change of miRNA concentrations, and the reproducibility of measurement was acceptable. The proposed method is potentially useful for simultaneous detection of multiple miRNAs with high sensitivity. [ABSTRACT FROM AUTHOR]

This is the first study to employ a reaction flow (RF) HPLC column of a short 5 cm length to identify antioxidants in a variety of complex samples represented by tea, utilising simultaneous multiplexed detection techniques. The detection responses included: underivatised UV at 280 nm, underivatised fluorescence detection (FLD) and a post column derivatisation (PCD) colorimetric response for antioxidants via the ferric reducing antioxidant power (FRAP) assay. Both similarities and differences in the chromatograms obtained using the three detection modes highlight the power of multiplexed detection. The main findings from the bioactive profiling include: the Fruit of the Forest tea contains the lowest level of antioxidants and is clearly distinct from the other three teas which are based on Camellia sinensis . The antioxidants detected in the green tea and the two black teas are similar. The green tea contains a higher concentration of the two main antioxidants that eluted at 4.0 and 5.7 min. The simplicity of sample analysis and chemical profiling using RF columns and selective detection was the focus of this study via the analysis of antioxidants in tea using the FRAP reagent. The main advantage of RF-PCD is the ability to split flows for simultaneous detection with minimal post column dead volume contributions, making peak matching between detectors much easier. To date, few studies exploit the power of RF-PCD multiplexed detection to differentiate and identify bioactive compounds in various complex samples. Applications of the approach developed in this study may include antioxidant screening and profiling of natural products, foods and beverages. [ABSTRACT FROM AUTHOR]

A novel approach for the analysis of phenolics in tea was developed using multiplexed simultaneous detection. This is the first study employing a short reaction flow (RF) chromatography column (length 50 mm) for the analysis of phenolics in complex samples (not a standard mixture of compounds). RF separation efficiency was high as the detectors are multiplexed simultaneously, rather than sequentially. The delay time between RF detection responses is minor (1.2 s) providing simple peak assignment between the phenolic detection and conventional detectors, such as the UV and fluorescence detectors (FLD). Thirty-nine phenolics were detected amongst the five different tea samples (three from Camellia sinensis and two herbal teas). The highest number of phenols detected was 24 for English Breakfast tea (four highly abundant), 19 for Earl Grey (six highly abundant), 13 in Pure Green (two highly abundant) and 9 in both Fruit of the Forest (two highly abundant) and Camomile (none highly abundant) herbal teas. Numerous low concentration phenolic compounds in the Fruit of the Forest tea were not included due to their abundance being only slightly above the noise level of the detection process. The relative response factor (RRF) analysed at 520 and 500 nm aided to identify unique phenolics to each sample with similar retention times; three phenolics were unique to the Pure Green tea (2.30, 4.11 and 5.80 min) and two to the Earl Grey Tea (4.11 and 5.80 min). The Fruit of the Forest tea and the Camomile tea were both distinctly different from all other teas. This approach may be used for phenolic profiling of food, beverages or natural products. [ABSTRACT FROM AUTHOR]

Herein, a highly-sensitive microRNA (miRNA) detection strategy was developed by combining bio-bar-code assay (BBA) with catalytic hairpin assembly (CHA). In the proposed system, two nanoprobes of magnetic nanoparticles functionalized with DNA probes (MNPs-DNA) and gold nanoparticles with numerous barcode DNA (AuNPs-DNA) were designed. In the presence of target miRNA, the MNP-DNA and AuNP-DNA hybridized with target miRNA to form a “sandwich” structure. After “sandwich” structures were separated from the solution by the magnetic field and dehybridized by high temperature, the barcode DNA sequences were released by dissolving AuNPs. The released barcode DNA sequences triggered the toehold strand displacement assembly of two hairpin probes, leading to recycle of barcode DNA sequences and producing numerous fluorescent CHA products for miRNA detection. Under the optimal experimental conditions, the proposed two-stage amplification system could sensitively detect target miRNA ranging from 10 pM to 10 aM with a limit of detection (LOD) down to 97.9 zM. It displayed good capability to discriminate single base and three bases mismatch due to the unique sandwich structure. Notably, it presented good feasibility for selective multiplexed detection of various combinations of synthetic miRNA sequences and miRNAs extracted from different cell lysates, which were in agreement with the traditional polymerase chain reaction analysis. The two-stage amplification strategy may be significant implication in the biological detection and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Aim: Development of a label-free multiplexed point-of-care diagnostic device for a panel of cardiac biomarkers - cardiac troponin-T (cTnT), troponin-I (cTnI) and B-type natriuretic peptide (BNP).Methods: A nonfaradaic electrochemical immunoassay designed with anisotropic high surface area ZnO nanostructures grown using low-temperature hydrothermal methods was selectively immobilized with capture antibodies. Multiplexed detection in human serum using ZnO nanostructures based on complementary electrochemical measurement techniques - electrochemical impedance spectroscopy and Mott-Schottky.Results: Linear signal response for detection of three biomarkers in human serum with dynamic range of 1 pg/ml-100 ng/ml and limit of detection at 1 pg/ml and low signal response to background interferences was achieved.Conclusion: First demonstration of simultaneous detection of three cardiac biomarkers in clinically relevant range with sensor's analytical performance and linear response of detection showed potential utility in screening clinical samples for early diagnosis of acute myocardial infarction and chronic heart failure. [ABSTRACT FROM AUTHOR]

In response to the urgent requirement for high-throughput multiplexed detection, a novel dual-encoded suspension array (SA) based on thickness and digital fluorescent peak is proposed, along with the corresponding optical system. The SAs are prepared by thickness encoded micro-glass chips (MGCs) with electrostatically adsorbed quantum dots (QDs). Using the thickness characteristics of microcarrier itself and digital fluorescence peak (DFP) as the encoded signals ensure the high encoding stability with simple fabrication process at a low cost. A spectral-domain phase and fluorescence spectroscopy microscopy (SDPFSM) system is designed for the first time to decode the thickness information and DFP with ultrahigh accuracy. The high selectivity and sensitivity of the dual-encoded SAs are verified by the multiplexed immunoassays. The quantitative detection of anti-human IgG, anti-mouse IgG, and anti-rabbit IgG was realized with the limits of detection (LOD) of 0.19, 0.35 and 0.31 pM/mL and the sensitivity of 713, 470 and 456 a.u./(pM/mL), respectively. The coding number increases by a factor of 458 compared with the traditional fluorescence coding method due to the additional use of the thickness information of the MGCs itself as a coding channel. The aforementioned properties demonstrate that the dual-encoded SA and the corresponding decoding platform are promising and robust in multiplexed detection. • An dual-encoded SA combining the MGCs and QDs is provided for multiplexed detection. • The thickness feature of microcarrier itself and DFP are used as the encoded signals. • The SA improves coding stability and codes number with simple process at a low cost. • The novel optical decoding system provides high accuracy and a wide dynamic range. [ABSTRACT FROM AUTHOR]

Using biosensor to screen for Alzheimer's disease (AD) facilitates early detection of AD with high sensitivity and accuracy. This approach overcomes the limitations of conventional AD diagnostic methods, such as neuropsychological assessment and neuroimaging analysis. Here, we propose a simultaneous analysis of signal combinations generated by four crucial AD biomarkers (Amyloid beta 1–40 (Aβ 40), Aβ 42 , total tau 441 (tTau 441), and phosphorylated tau 181 (pTau 181)) by inducing a dielectrophoretic (DEP) force on fabricated interdigitated microelectrode (IME) sensor. By applying an optimal DEP force, our biosensor selectively concentrates and filters the plasma-based AD biomarkers, exhibiting high sensitivity (limit of detection <100 fM) and selectivity in the plasma-based AD biomarkers detection (p < 0.0001). Consequently, it is demonstrated that a complex combined signal comprising four AD-specific biomarker signals (Aβ 40 − Aβ 42 + tTau 441 − pTau 181) can differentiate between patients with AD and healthy subjects with high accuracy (78.85%) and precision (80.95%) (p < 0.0001). [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are recognized as potential biomarkers for the early diagnosis and prognosis of different diseases. Multiplexed and accurate miRNA quantification methods with equivalent detection efficiency are particularly crucial due to their complex biological functions and lack of a unified internal reference gene. Here, a unique multiplexed miRNA detection method, named Specific Terminal-Mediated miRNA PCR (STEM-Mi-PCR), was developed. It mainly includes a linear reverse transcription step using tailored-designed target specific capture primers, followed by an exponential amplification process using two universal primers to execute the multiplex assay. For proof of concept, four miRNAs were used as models to develop a multiplexed detection assay within one tube simultaneously and then evaluate the performance of the established STEM-Mi-PCR. The sensitivity of the 4-plexed assay was approximately 100 aM with an equivalent amplification efficiency (95.67 ± 8.58%), and had no cross-reactivity each other with high specificity. Quantification of different miRNAs in twenty patients' tissues shown variation from approximately pM to fM concentration level, demonstrating the possibility of practical application of the established method. Moreover, this method was extraordinarily capable of single nucleotide mutation discrimination in different let-7 family members with no more than 0.7% nonspecific detection signal. Hence, the STEM-Mi-PCR we proposed here paves an easy and promising way for miRNA profiling in future clinical applications. [Display omitted] • A multiple, equivalent, and accurate miRNA detection method is proposed. • The STEM-Mi-PCR realizes a 4-plex detection with equivalent amplification efficiency. • The strategy achieves high specificity with no more than 0.7% nonspecific signal. [ABSTRACT FROM AUTHOR]

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope-labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am-tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am-tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am-tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO4 concentrations. [ABSTRACT FROM AUTHOR]

A universal aptameric system based on the taking advantage of double-stranded DNA/perylene diimide (dsDNA/PDI) as the signal probe was developed for multiplexed detection of small molecules. Aptamers are single-stranded DNA or RNA oligonucleotides which are selected in vitro by a process known as systematic evolution of ligands by exponential enrichment. In this work, we synthesized a new kind of PDI and reported this aggregated PDI could quench the double-stranded DNA (dsDNA)-labeled fluorophores with a high quenching efficiency. The quenching efficiencies on the fluorescence of FAM, TAMRA and Cy5 could reach to 98.3%±0.9%, 97.2%±0.6% and 98.1%±1.1%, respectively. This broad-spectrum quencher was then adopted to construct a multicolor biosensor via a label-free approach. A structure-switching-triggered enzymatic recycling amplification was employed for signal amplification. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity towards small analytes. For other targets, changing the corresponding aptamer can achieve the goal. The quencher did not interfere with the catalytic activity of nuclease. The biosensor could be manipulated with similar sensitivity no matter in pre-addition or post-addition manner. Moreover, simultaneous and multiplexed analysis of several small molecules in homogeneous solution was achieved, demonstrating its potential application in the rapid screening of multiple biotargets. [ABSTRACT FROM AUTHOR]

In this work, a real-time assay for highly sensitive, label-free, multiplexed electrical detection of lung cancer biomarkers was developed by using silicon nanowire field-effect (SiNW-FET) devices. Highly responsive SiNW arrays were fabricated using a CMOS-compatible anisotropic self-stop etching technique with mass reproducibility and low cost character. The SiNW nanosensor was integrated with PDMS microfluidic device, which allows rapid analyte delivery, makes the analysis to be conducted using exceedingly small samples and enables potential multiplexed detection. The nanowire arrays allowed highly selective and sensitive multiplexed detection of microRNA (miRNA)−126 and CEA. Due to high surface-to-volume ratio that the nanowire dimensions confer, the detection floor of single molecule was achieved. The potential utility in identifying clinical samples for early diagnosis of cancer was demonstrated by analyzing biomarkers in clinical related samples. The developed nanosensor with capability for multiplexed real-time monitoring of biomarkers with high sensitivity and selectivity in clinically relevant samples is highly attractive for diagnosis and treatment of cancer and other diseases. [ABSTRACT FROM AUTHOR]

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reductive amination to evaluate the multiplexing capacity of luminescence resonance energy transfer (LRET) between UCNPs and quantum dots (QDs). This is the first account of a multiplexed bioassay strategy that demonstrates the principle of use of a single form of UCNP as donor and three different color emitting QDs as acceptors to concurrently determine three analytes. Broad absorbance profiles of green, orange and red QDs that spanned from the first exciton absorption peak to the UV region were in overlap with a blue emission band from UCNPs composed of NaYF 4 that was doped with 30% Yb 3+ , 0.5% Tm 3+ , allowing for LRET that was stimulated using 980 nm near-infrared radiation. The characteristic narrow and well-defined emission peaks of UCNPs and QDs allowed for the collection of luminescence from each nanoparticle using a band-pass optical filter and an epi-fluorescence microscope. The LRET system was used for the concurrent detection of uidA, Stx1A and tetA gene fragments with selectivity even in serum samples, and reached limits of detection of 26 fmol, 56 fmol and 76 fmol, respectively. [ABSTRACT FROM AUTHOR]

Multiplexed detection of protein biomarkers offers new opportunities for early diagnosis and efficient treatment of complex diseases. Cardiovascular diseases (CVDs) has the highest mortality risk in USA and Europe with 15–20 million cases being reported annually. Cardiac Troponins (T and I) are well established protein biomarkers associated with heart muscle damage and point-of-care monitoring of both these two biomarkers has significant benefits on patient care. A flexible disposable electrochemical biosensor device comprising of vertically oriented zinc oxide (ZnO) nanostructures was developed for rapid and simultaneous screening of cardiac Troponin-I (cTnI) and cardiac-Troponin-T (cTnT) in a point-of-care sensor format. The biosensors were designed by selective hydrothermal growth of ZnO nanostructures onto the working electrodes of polyimide printed circuit board platforms, resulting in the generation of high density nanostructure ZnO arrays based electrodes. The size, density and surface terminations of the nanostructures were leveraged towards achieving surface confinement of the target cTnT and cTnI molecules on to the electrode surface. Multiplexing and simultaneous detection was achieved through sensor platform design comprising of arrays of Troponin functionalized ZnO nanostructure electrodes. The sensitivity and specificity of the biosensor was characterized using two types of electrochemical techniques; electrochemical impedance spectroscopy (EIS) and Mott-Schottky analysis on the same sensor platform to demonstrate multi-configurable modes. Limit of detection of 1 pg/mL in human serum was achieved for both cTnI and cTnT. Cross reactivity analysis showed the selectivity of detecting cTnT and cTnI in human serum with wide dynamic range. [ABSTRACT FROM AUTHOR]

The authors report on a rapid voltammetric method for simultaneous determination of the pathogens E. coli and Salmonella typhimurium ( S. typh.) by detecting the rfbE gene of E. coli O157:H7 and gyrB gene of S. typh., respectively, and by using polymerase-assisted target recycling amplification. The assay was constructed by self-assembly of the respective hairpin probes (labeled with the electrochemical probes Methylene Blue and ferrocene) on the surface of a gold electrode. After hybridization between target DNA and hairpin probes (HPs) has occurred, the primers hybridize with the open-chain HPs and initiate extension reactions in the presence of polymerase and deoxyribonucleoside triphosphates. This results in the release of the redox labels from the electrode surface and the target dissociating from the HPs. The released target will bind to other HPs to activate new cycles, which results in enhanced suppression of current, measured best at −0.27 V and +0.36 V (vs. Ag/AgCl) for parallel detection of E. coli DNA and S. typh. DNA, respectively. The method presented here based on target recycling amplification and its integration into multiplexed electrochemical detection of pathogens was successfully applied to quantitative determination of E. coli O157:H7 and S. typh. in synthetic samples. In our perception, the strategy presented here represents a rapid and universal platform for sensitive and multiplexed quantitation of pathogens and related molecular diagnostic targets of relevance in food safety control. [ABSTRACT FROM AUTHOR]

This work describes the use of mass-produced stainless-steel pins as low-cost electrodes to develop simple and portable amperometric glucose biosensors. A potentiostatic three-electrode configuration device is designed using two bare pins as reference and counter electrodes, and a carbon-ink coated pin as working electrode. Conventional transparency film without any pretreatment is used to punch the pins and contain the measurement solution. The interface to the potentiostat is very simple since it is based on a commercial female connection. This electrochemical system is applied to glucose determination using a bienzymatic sensor phase (glucose oxidase/horseradish peroxidase) with ferrocyanide as electron-transfer mediator, achieving a linear range from 0.05 to 1 mM. It shows analytical characteristics comparable to glucose sensors previously reported using conventional electrodes, and its application for real food samples provides good results. The easy modification of the position of the pins allows designing different configurations with possibility of performing simultaneous measurements. This is demonstrated through a specific design that includes four pin working-electrodes. Different concentrations of antibody labeled with alkaline phosphatase are immobilized on the pin-heads and after enzymatic conversion of 3-indoxylphosphate and silver nitrate, metallic silver is determined by anodic stripping voltammetry. [ABSTRACT FROM AUTHOR]

Reaction flow (RF) chromatography with fluorescamine reagent and fluorescence detection (FLD) was used for the analysis of amino acids. The performance of RF chromatography was tested against several optimized conventional postcolumn derivatization (PCD) methods. RF columns achieved greater sensitivity compared to conventional PCD methods, without the need for reaction loops, which resulted in more efficient separations. The RF-PCD method also achieved limits of detection (LOD) from the low picomole to subnanomole range. The calibration data of the RF-PCD technique yieldedR2 ≥ 0.99 and % relative standard deviation in peak areas ranging from 0.34% to 5%. Through reaction flow chromatography, multiplexed detection was also achieved allowing the monitoring and analysis of derivatized and nonderivatized flow streams simultaneously. [ABSTRACT FROM AUTHOR]

Early detection is often the key to successful treatment and patient survival. The presence of various disease signaling salivary biomarkers that accurately reflect normal and disease states in humans as well as the sampling benefits and easy storage and transport of saliva compared to blood are some of the reasons for being increasingly recognized as an attractive diagnostic fluid. This explains the burgeoning research field in developing new methodologies able to determine low concentrations of different levels of biomarkers in saliva in a simple and rapid way. Aiming at overcoming some limitations of conventional strategies, electrochemical affinity biosensors have demonstrated to offer interesting alternatives for salivary diagnostics. This paper reviews briefly the significance of saliva and salivary biomarkers for clinical diagnosis and therapeutic applications, and highlights recent advances, main challenges and future prospects in sensing bioaffinity platforms developed for the single or multiplexed determination of salivary biomarkers. [ABSTRACT FROM AUTHOR]

We have developed a novel graphene oxide (GO) sensing platform based on enzymatic phosphorylation for the multiplexed detection of protein kinases. The dye-labeled peptide substrate exhibits strong fluorescence in the absence of kinases. When the biotinylated adenosine 5′-triphosphate co-substrate and kinase are present, the peptide substrate is phosphorylated by kinase, and the phosphorylated peptide product carrying both the biotin site and the dye can be assembled onto streptavidin-coated GO via streptavidin-biotin chemistry, leading to fluorescence quenching. Importantly, since GO is a highly efficient quencher for multiple different fluorophores, the platform allows simultaneous detection of multiple protein kinases using different peptide substrates labeled with corresponding dyes. As a proof-of-concept, we demonstrate that this GO sensing platform can simultaneously and selectively detect protein kinase A, protein kinase Abl and protein kinase Src with low detection limits of 0.005 U/mL, 0.02 U/mL and 0.05 U/mL, respectively. Furthermore, the application of this method for detection of multiple protein kinases in biological samples and protein kinase inhibitor screening has also been demonstrated. This approach holds great promise as a routine tool for the high-throughput screening of protein kinases and their inhibitors. [ABSTRACT FROM AUTHOR]

Foodborne diseases caused by pathogens may threaten public health and the social economy. We demonstrated a method for identifying pathogenic Listeria monocytogenes using DNA logic operations. To achieve accurate species distinguishing, three specific sequences of Listeria monocytogenes genomic DNA were screened out and used as the feature sequences. Three complementary probes with tag modification were designed as sensing elements and exert affinity for magnetic beads, glucose oxidase (GOx), and horseradish peroxidase (HRP). To obtain a digital output (YES/NO answer) for rapid determination, a Boolean logic function was employed. Three sensing probes enabled the recognition of the target sequence (input) and the formation of a target DNA/probe hybrid. Through magnetic separation and affinity binding events, the target DNA/probes hybrid led to the construction of GOx/HRP enzyme cascade, which produced a visualized color signal (output) in the presence of substrates, glucose, and 3, 3′, 5, 5′-tetramethylbenzidine (TMB). A hybridization chain reaction (HCR) was coupled with this sensing scaffold to increase the binding of the enzyme cascade and amplify the output signal. The logical functional biosensor showed high selectivity of Listeria monocytogenes over other Listeria species. This sensing platform provides a simple, sensitive, and highly specific method for detecting Listeria monocytogenes. [Display omitted] • A method for identifying foodborne pathogens using DNA logic operations. • Genomic DNA was screened and used as the feature sequences for specific detection. • Enzyme cascade and hybridization chain reactions yield and amplify output signals. [ABSTRACT FROM AUTHOR]

Health initiatives worldwide demand affordable point-of-care devices to aid in the reduction of morbidity and mortality rates of high-incidence infectious and noncommunicable diseases. However, the production of robust and reliable easy-to-use diagnostic platforms showing the ability to quantitatively measure several biomarkers in physiological fluids and that could in turn be decentralized to reach any relevant environment remains a challenge. Here, we show the particular combination of paper-microfluidic technology, electrochemical transduction, and magnetic nanoparticle-based immunoassay approaches to produce a unique, compact, and easily deployable multiplex device to simultaneously measure interleukin-8, tumor necrosis factor-α, and myeloperoxidase biomarkers in sputum, developed with the aim of facilitating the timely detection of acute exacerbations of chronic obstructive pulmonary disease. The device incorporates an on-chip electrochemical cell array and a multichannel paper component, engineered to be easily aligned into a polymeric cartridge and exchanged if necessary. Calibration curves at clinically relevant biomarker concentration ranges are produced in buffer and artificial sputum. The analysis of sputum samples of healthy individuals and acutely exacerbated patients produces statistically significant biomarker concentration differences between the two studied groups. The device can be mass-produced at a low cost, being an easily adaptable platform for measuring other disease-related target biomarkers.

Nanopores are versatile single-molecule sensors that are being used to sense increasingly complex mixtures of structured molecules with applications in molecular data storage and disease biomarker detection. However, increased molecular complexity presents additional challenges to the analysis of nanopore data, including more translocation events being rejected for not matching an expected signal structure and a greater risk of selection bias entering this event curation process. To highlight these challenges, here, we present the analysis of a model molecular system consisting of a nanostructured DNA molecule attached to a linear DNA carrier. We make use of recent advances in the event segmentation capabilities of Nanolyzer, a graphical analysis tool provided for nanopore event fitting, and describe approaches to the event substructure analysis. In the process, we identify and discuss important sources of selection bias that emerge in the analysis of this molecular system and consider the complicating effects of molecular conformation and variable experimental conditions (e.g., pore diameter). We then present additional refinements to existing analysis techniques, allowing for improved separation of multiplexed samples, fewer translocation events rejected as false negatives, and a wider range of experimental conditions for which accurate molecular information can be extracted. Increasing the coverage of analyzed events within nanopore data is not only important for characterizing complex molecular samples with high fidelity but is also becoming essential to the generation of accurate, unbiased training data as machine-learning approaches to data analysis and event identification continue to increase in prevalence.

Digital immunoassays with multiplexed capacity, ultrahigh sensitivity, and broad affordability are urgently required in clinical diagnosis, food safety, and environmental monitoring. In this work, a multidimensional digital immunoassay has been developed through microparticle-based encoding and artificial intelligence-based decoding, enabling multiplexed detection with high sensitivity and convenient operation. The information encoded in the features of microspheres, including their size, number, and color, allows for the simultaneous identification and accurate quantification of multiple targets. Computer vision-based artificial intelligence can analyze the microscopy images for information decoding and output identification results visually. Moreover, the optical microscopy imaging can be well integrated with the microfluidic platform, allowing for encoding-decoding through the computer vision-based artificial intelligence. This microfluidic digital immunoassay can simultaneously analyze multiple inflammatory markers and antibiotics within 30 min with high sensitivity and a broad detection range from pg/mL to μg/mL, which holds great promise as an intelligent bioassay for next-generation multiplexed biosensing.

The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green-red-yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.

This work reports a portable, origami-type paper device with a plasmonic fluor-labeled microneedle sensing module for the multiplexed quantification of anemia biomarkers in whole blood. Sequential steps, including serum separation, target enrichment, and multiplexed readout by a gel imager, are rapidly accomplished with the flexible and highly integrated device. The microneedle array enabled efficient sampling of trace targets from ng mL -1 to pg mL -1 level. Combined with the plasmonic fluor label, the signal is improved by ≈7.6 folds compared with the flat substrate-based assay. The device is applied to simultaneously quantify hemoglobin (Hb), ferritin, folic acid (FA), and vitamin B 12 (VB 12 ), which are four anemia biomarkers distributed in different environments with different concentration ranges. Featured by the small sample volume (150 µL), short assay time (20 min), low cost (2 $), robust stability, and user-friendliness, the device is promising for the rapid and accurate diagnosis of anemia in real practice., (© 2023 Wiley-VCH GmbH.)

A rapid and multiplexed immunosensor was developed based on a quantum dot (QD)-reverse assaying strategy (RAS) and immuno-magnetic beads (IMBs) for one-step and simultaneous detection of Escherichia coli O157: H7 and Salmonella . In a conventional QD-based immunosensor, the fluorescence signal of the “IMBs-target-QD” immunoconjugate is directly used as the assaying readout. However, the fluorescence signal is affected by IMBs due to light scattering and the “IMBs-target-QD” immunoconjugate needs multiple washing and re-suspension steps. To address these problems, we use the surplus QD-antibody conjugate as signal readout in the RAS, which prevents interference from the IMBs, increases the fluorescence signal, and avoids complex operations. Compared with conventional QD-based immunosensor, the sensitivity of QD-RSA immunosensor for detection of Escherichia coli O157: H7 has been improved fifty-fold, and whole analysis procedure can be finished within 1 h. Therefore, this RSA strategy is promising for improving the performance of QD-based immunosensors and could greatly broaden their applications. [ABSTRACT FROM AUTHOR]

A multiplexed microfluidic biosensor made of poly(methylmethacrylate) (PMMA) was integrated into an array of organic blend heterojunction photodiodes (OPDs) for chemiluminescent detection of pathogens. Waterborne Escherichia coli O157:H7, Campylobacter jejuni and adenovirus were targeted in the PMMA chip, and detection of captured pathogens was conducted by poly(2,7-carbazole)/fullerene OPDs which showed a responsivity over 0.20 A/W at 425 nm. The limits of chemiluminescent detection were 5 × 105 cells/mL for E. coli, 1 × 105 cells/mL for C. jejuni, and 1 × 10−8 mg/mL for adenovirus. Parallel analysis for all three analytes in less than 35 min was demonstrated. Further recovery tests illustrated the potential of the integrated biosensor for detecting bacteria in real water samples.

The article reviews the current status of label-free integrated optical biosensors focusing on the evolution over the years of their analytical performance. At first, a short introduction to the evanescent wave optics is provided followed by detailed description of the main categories of label-free optical biosensors, including sensors based on surface plasmon resonance (SPR), grating couplers, photonic crystals, ring resonators, and interferometric transducers. For each type of biosensor, the detection principle is first provided followed by description of the different transducer configurations so far developed and their performance as biosensors. Finally, a short discussion about the current limitations and future perspectives of integrated label-free optical biosensors is provided.

Paper-based lateral flow devices (LFDs) are regarded as ideal low-cost diagnostic solutions for point-of-care (POC) scenarios that allow rapid detection of a single analyte within a fluidic sample, and have been in common use for a decade. In recent years, there has been an increasing need for rapid and simultaneous detection of multiple analytes present within a single sample and to facilitate this, we report here a novel solution—detection using a multi-path LFD created via the precise partitioning of the single flow-path of a standard LFD using our previously reported laser direct-write (LDW) technique. The multiple flow-paths allow the simultaneous detection of the different analytes individually within each of the parallel channels without any cross-reactivity. The appearance of coloured test lines in individual channels indicates the presence of the different analytes within a sample. We successfully present the use of a LDW-patterned multi-path LFD for multiplexed detection of a biomarker panel comprising C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), used for the diagnosis of bacterial infections. Overall, we demonstrate the use of our LDW technique in the creation of a novel LFD that enables multiplexed detection of two inflammation markers within a single LFD providing a detection protocol that is comparatively more efficient than the standard sequential multiplexing procedure.

Molecular diagnostics have significantly advanced the early detection of diseases, where the electrochemical sensing of biomarkers (e.g., DNA, RNA, proteins) using multiple electrode arrays (MEAs) has shown considerable promise. Nanostructuring the electrode surface results in higher surface coverage of capture probes and more favorable orientation, as well as transport phenomena unique to nanoscale, ultimately leading to enhanced sensor performance. The central goal of this study is to investigate the influence of electrode nanostructure on electrically-guided immobilization of DNA probes for nucleic acid detection in a multiplexed format. To that end, we used nanoporous gold (np-Au) electrodes that reduced the limit of detection (LOD) for DNA targets by two orders of magnitude compared to their planar counterparts, where the LOD was further improved by an additional order of magnitude after reducing the electrode diameter. The reduced electrode diameter also made it possible to create a np-Au MEA encapsulated in a microfluidic channel. The electro-grafting reduced the necessary incubation time to immobilize DNA probes into the porous electrodes down to 10 min (25-fold reduction compared to passive immobilization) and allowed for grafting a different DNA probe sequence onto each electrode in the array. The resulting platform was successfully used for the multiplexed detection of three different biomarker genes relevant to breast cancer diagnosis.

Foodborne pathogens have raised significant concerns in human public health. Rapid, high-sensitive, low-cost, and easy-to-use testing methods for food safety are needed. In this study, we developed a finger-actuated microfluidic biosensor (FA-MB) for multiplexed detection of Bacillus cereus and other six common foodborne pathogens based on one-pot CRISPR/Cas12a combined with recombinase aided amplification (RAA). Wells for RAA and CRISPR/Cas12a were isolated to avoid interference, while finger-actuated one-way control valves were incorporated to fulfill the unidirectional flow of RAA products to the CRISPR/Cas12a reaction wells, realizing one-pot RAA-CRISPR/Cas12a assay. The final fluorescent signal was acquired and processed by a smartphone. Under selected experimental conditions, seven pathogenic bacteria could be tested in about 1 h with the limits of detection (LODs) below 500 CFU/mL. Recoveries ranged from 90% to 116% of the spiked samples were readily achieved. The proposed FA-MB is highly integrated and easy-to-use, and could be used for rapid, high-sensitive point of care (POC) testing without the external driving device, suitable for resource-constrained settings. [Display omitted] [ABSTRACT FROM AUTHOR]

Multi-channel evaluation on a single sensor interface is highly valuable but full of challenge. Herein, a universal light-addressable electrochemical immunoassay based on silicon photoelectrodes was proposed for spatially-resolved detection of prostate-specific antigen (PSA) as a model target. To stabilize and functionalize silicon substrate, a simple and robust antifouling coating was prepared by cross-linking bovine serum albumin (BSA) matrix with glutaraldehyde (GA). For the PSA detection, an electrochemical sandwich-type immunosensor was performed on the basis of BSA@GA functionalized silicon, in which horse radish peroxidase (HRP) was used to catalyze the reaction of H 2 O 2 and hydroquinone (HQ), resulting in in situ generation of benzoquinone (BQ). The light-induced local reduction photocurrent of BQ was then recorded as the sensor response by irradiating the target spot of photoelectrode respectively with a moveable light pointer. The results demonstrated the feasibility of localized measurements of enzymatic reactions. Coupling with light-addressable electrochemistry (LAE) and enzyme-linked immunosorbent assay (ELISA) protocol, multiplexed PSA detection could be achieved on a single sensor chip with good sensitivity and specificity. This proof-of-concept study provides a promising platform for high-throughput and accurate immunoanalysis in diagnostic or biodefense applications. • Light-addressable electrochemical immunoassay for detection of antigen. • The integration of ELISA into light-addressable electrochemistry. • The localized measurements of enzymatic reaction on silicon photoelectrode. • Multiplexed PSA detection was achieved on a single sensor interface. [ABSTRACT FROM AUTHOR]

Nanozymes with enzyme-mimicking catalytic activity and unique functions have stimulated increasing interest in the biosensing field. Herein, we report a magnetic nanozyme (MNE) with integrated superior peroxidase-like activity and efficient mixing ability. This nanozymatic magnetic nanomixer is synthesized by depositing a Fe2+-doped polydopamine coating on the surface of well-aligned magnetic nanoparticles to form a rigid chain-like nanostructure. Polydopamine coating of the nanozymatic MNE allows for efficient immobilization of natural enzymes such as glucose oxidase, cholesterol oxidase or urate oxidase to produce a series of enzymes-immobilized MNE (MNE@enzymes) with intrinsic multienzyme cascade properties. These MNE@enzymes show synchronously rotating capability in spinning magnetic fields, which leads to an 80∼100% improvement in their overall catalytic efficiencies. In the on-chip detection of small molecular metabolites (i.e., glucose, cholesterol, and uric acid), the rotating MNE@enzymes lead to detection sensitivities 2.1∼4.3 times higher than those of the static ones. Importantly, the consistent performance of the rotating MNE@enzymes offers the possibility of integrating the detection of glucose, free cholesterol and uric acid into a single multiplexing microchip assay with smartphone readout, affording an improved sensitivity, good selectivity and reliability. The designed enzymes-loaded MNEs holds great promise in developing rapid and ultrasensitive measurements of diverse targets of healthcare concerns using portable devices. [Display omitted] [ABSTRACT FROM AUTHOR]