Sandwich Format Research Articles

Rapid and sensitive pathogen detection platform based on a lanthanide-labeled immunochromatographic strip test combined with immunomagnetic separation

Sensitive point-of-care pathogen detection methods are urgently required as pathogens pose a significant threat to food safety and human health. Here, we reported a platform based on a lanthanide-labeled immunochromatographic strip test combined with immunomagnetic separation for the detection of pathogens in food and clinical samples. First, immunomagnetic separation technology was employed as a pre-treatment to eliminate the effect of background in the samples. Then, the lanthanide chelate-loaded carboxyl-modified polystyrene nanoparticles, exhibiting bright luminescence, wide Stokes shift, and excellent stability, were prepared and used to label pathogens with antibody. Finally, an immunochromatographic strip test, based on a sandwich format and supported by our custom-designed portable reader, was used to rapidly quantify the result. Using Salmonella typhimurium as a model pathogen, under optimal conditions, we achieved a detection limit of 103 CFU/mL and a linear range of 104–106 CFU/mL in milk and human serum with good recovery, reproducibility, and specificity. Compared with the existing colloidal gold-based immunochromatographic strip test, our platform showed high sensitivity (100-fold improvement) and provided quantitative determination. Our detection platform was therefore demonstrated to be highly sensitive, rapid (within 1.5 h), and cost-effective, providing a promising platform for point-of-care pathogen detection in food and clinical fields.

Lateral Flow Immunoassay to Detect the Addition of Beef, Pork, Lamb, and Horse Muscles in Raw Meat Mixtures and Finished Meat Products.

A lateral flow immunoassay for sensitive detection of skeletal troponin I (TnI) as a specific, thermostable marker of muscle tissue was developed. Due to the antibodies’ choice, the assay specifically detects mammalian TnI (in beef, pork, lamb, and horse) but does not detect bird TnI (in chicken or turkey), thus enabling differentiation of these types of raw meat materials. The assay is based on a sandwich format of the analysis using gold nanoparticles as labels. The time of the assay is 15 min, and the TnI detection limit is 25 ng/mL. A buffer solution is proposed for efficient extraction of TnI from muscle tissues and from finished meat products that have undergone technological processing (smoking–cooking–smoking, cooking and smoking). The possibility of detecting beef addition in minced chicken down to 1% was demonstrated.

Development of a specific troponin I detection system with enhanced immune sensitivity using a single monoclonal antibody.

Using an immunoassay in combination with surface plasmon fluorescence spectroscopy (SPFS), we report the rapid detection of troponin I, a valuable biomarker for diagnosis of myocardial infarction. We discuss the implementation of (i) direct, (ii) sandwich, and (iii) competitive assay formats, based on surface plasmon resonance and SPFS. To elucidate the results, we relate the experiments to orientation-dependent interaction of troponin I epitopes with respective immunoglobulin G antibodies. A limit of detection (LoD) of 19 pM, with 45 min readout time, was achieved using single monoclonal antibody that is specific for one epitope. The borderline between normal people and patients is 20 pM to 83 pM cTnI concentration, and upon the outbreak of acute myocardial infraction it can raise to 2 nM and levels at 20 nM for 6–8 days, therefore the achieved LoD covers most of the clinically relevant range. In addition, this system allows for the detection of troponin I using a single specific monoclonal antibody, which is highly beneficial in case of detection in real samples, where the protein has a complex form leading to hidden epitopes, thus paving the way towards a system that can improve early-stage screening of heart attacks.

Development of an Aptamer-Based Lateral Flow Assay for the Detection of C-Reactive Protein Using Microarray Technology as a Prescreening Platform.

For improved cost-effectiveness and temperature-stability, a ready to use lateral flow assay (LFA) is developed in this work for detecting inflammation/infection biomarker C-reactive protein (CRP) in human patient samples on the basis of aptamers. In prescreening investigations, an aptamer with CRP affinity was immobilized on microarray chips in forward and sandwich formats to optimize assay conditions. We suggest these microarray techniques as a resource-sparing and fast-screening instrument for evaluation of various conditions. The capability of the aptamer to detect CRP was shown. Optimized assay conditions were consequently transferred to the LFA-platform. Here we could demonstrate for the first time an aptamer-based LFA for the detection of CRP in human patient samples in pathologically relevant concentrations. The cutoff for CRP detection is set at 10 mg/L, providing a distinctive "yes" (≥10 mg/L CRP) or "no" (<10 mg/L CRP) answer for the patient. The resulting aptamer-based LFA is promising with regard to its application as point-of-care testing (POCT) for efficient monitoring, especially of patients affected by frequent infections or inflammations.

Mild reduction-promoted sandwich aptasensing for simple and versatile detection of protein biomarkers

Sandwich format has drawn considerable attention in aptasensing in view of simple design, convenient and stable application, and easy coupling with signal amplification strategies; however, developing sandwich aptasensing methods is still problematic, because it is a rather challenging and troublesome task to find or screen suitable aptamer pairs. Herein, we made use of mild reduction to propose a novel sandwich electrochemical aptasensing method for protein biomarker detection that avoided the need of aptamer pairs. In our design, target protein was captured by surface-immobilized aptamer and exposed active thiol groups after mild reduction. Then, maleimide-functionalized Probe DNA was linked to protein through Michael addition between maleimide and active thiol groups, thus forming a sandwich structure that could be used for signal output. Moreover, considering that the sequence of Probe DNA was freely designed, DNA-based amplification strategies could be easily incorporated in the method so as to further improve the detection sensitivity. Hybridization chain reaction was utilized here as an example, which could accumulate signaling molecules and finally arouse amplified electrochemical responses. Results demonstrated improved sensitivity and specificity of our method even in a complex serum samples by using α-fetoprotein as a model, and also revealed the versatility of our method by using both α-fetoprotein and Golgi protein 73 as examples. Our method might pave a feasible way for constructing sandwich aptasensing methods, which would provide a new insight into high-throughput determination of disease-related protein biomarkers in the future.

G-quadruplex 2′-F-modified RNA aptamers targeting hemoglobin: Structure studies and colorimetric assays

Biosensors that rely on aptamers as analyte-recognizing elements (also known as aptasensors) are gaining in popularity during recent years for analytical and biomedical applications. Among them, colorimetric ELISA-like systems seem very promising for biomarker detection in medical diagnostics. For their development, one should thoroughly consider the characteristics of the aptamers, with a particular focus on the secondary structure. In this study, we performed an in-depth structural study of previously selected hemoglobin-binding 2′-F-RNA aptamers using CD spectroscopy, enzymatic probing, and specific fluorophore binding. Only a combination of different assays allowed us to prove G-quadruplex formation for anti-hemoglobin 2′-F-RNA aptamers. We also demonstrated a possible application of these 2′-F-RNA aptamers for microplate colorimetric detection of human hemoglobin in both direct and sandwich formats.

Electrochemical genosensor for the detection of Alexandrium minutum dinoflagellates

This work addresses the development of a disposable electrochemical genosensor for the detection of the toxic dinoflagellate, Alexandrium minutum. Analyzing public databases, a specific 70 bp DNA probe, targeting A. minutum, was selected and designed. The genosensor methodology implied the immobilization of a A. minutum-specific DNA-capture probe onto screen-printed gold electrodes (SPGE). To improve both the selectivity and to avoid strong secondary structures, that could hinder the hybridization efficiency, a sandwich format of the A. minutum gene was designed using a fluorescein isothiocyanate-labelled signaling DNA probe and enzymatic amplification of the electrochemical signal.Using this electrochemical genosensor, a concentration range from 0.12 to 1.0 nM, a LD of 24.78 pM with a RSD <5.2% was determined. The genosensor was successfully applied to the selective analysis of the targeted A. minutum specific region denatured genomic DNA extracted from toxic dinoflagellates present in the Atlantic Ocean.

A rapid and sensitive lateral flow immunoassay (LFIA) test for the on-site detection of banana bract mosaic virus in banana plants

Banana bract mosaic virus (BBrMV) is a serious pathogen threatening the cultivation of banana and plantain worldwide. This study reports the development of a practical, rapid, sensitive, specific and user-friendly lateral flow immunoassay (LFIA) test for the on-site detection of BBrMV. The BBrMV coat protein (CP) was expressed in Escherichia coli and purified and used to immunize rabbits to produce a polyclonal antiserum (anti-BBrMVCP). The test was based on a double-antibody sandwich format. Protein-A affinity column-purified anti-BBrMVCP Immunoglobulins (IgG) (16 μg/mL), conjugated to ∼30 nm gold nanoparticles, was applied onto the conjugate pad. The anti-BBrMVCP IgG and goat anti-rabbit IgG were printed on the surface of a nitrocellulose filter membrane as the test line and control line, respectively. A positive result could be confirmed visually by the presence of a pink band that developed on the LFIA strip within 5−10 min. The detection limit of the test was 10 ng of the expressed recombinant BBrMV CP (rBBrMVCP), and a 1:20 dilution of the BBrMV-infected crude extract. This LFIA test was validated using 114 banana leaf samples randomly collected from the field and the results indicated a very high diagnostic sensitivity (99.04 %) and specificity (100 %) for the test. A Cohen's kappa coefficient of 0.861 obtained also indicated a very good agreement between the LFIA developed in this study and ELISA. This assay could be adopted by farmers, tissue culture industries and quarantine departments for surveys and surveillance. This is the first report on the development of a LFIA-based test for BBrMV detection.

Label-Free Immunoassay of Carcinoembryonic Antigen by Microfluidic Channel Biosensor Based on Imaging Ellipsometry and Its Clinic Application

This study represents a label-free immunoassay for carcinoembryonic antigen (CEA) by microfluidic biosensor based on imaging ellipsometry (BIE) and its application is further validated by clinic sera. The sandwich format of analysis configuration was adopted, in which Rat-mAb was served as capture antibody, rabbit anti-CEA polyclonal antibody (rabbit-pAb) as detection antibody and goat anti-rabbit IgG (goat-IgG) as an enhancer added to increase detection signal. The methodology includes rat-mAb concentration screening, rabbit-pAb and goat-IgG concentration optimization, creation of dose-response curve and determination of cutoff value. Results show the linear range is from 4.0–64.0 ng/ml with a lower limit of 1.0 ng/ml (S/N = 3). Application of the BIE to clinic samples demonstrated that the relation coefficient is 0.94 and substantial agreement with the results of ECLIA’s (Kappa = 0.733, U = 4.35 > U0.01). The proposed method exhibits the potential for application.

Magnetic Bead-Based Electrochemical Immunoassays On-Drop and On-Chip for Procalcitonin Determination: Disposable Tools for Clinical Sepsis Diagnosis.

Procalcitonin (PCT) is a known protein biomarker clinically used for the early stages of sepsis diagnosis and therapy guidance. For its reliable determination, sandwich format magnetic bead-based immunoassays with two different electrochemical detection approaches are described: (i) disposable screen-printed carbon electrodes (SPE-C, on-drop detection); (ii) electro-kinetically driven microfluidic chips with integrated Au electrodes (EMC-Au, on-chip detection). Both approaches exhibited enough sensitivity (limit of detection (LOD) of 0.1 and 0.04 ng mL−1 for SPE-C and EMC-Au, respectively; cutoff 0.5 ng mL−1), an adequate working range for the clinically relevant concentrations (0.5–1000 and 0.1–20 ng mL−1 for SPE-C and EMC-Au, respectively), and good precision (RSD < 9%), using low sample volumes (25 µL) with total assay times less than 20 min. The suitability of both approaches was successfully demonstrated by the analysis of human serum and plasma samples, for which good recoveries were obtained (89–120%). Furthermore, the EMC-Au approach enabled the easy automation of the process, constituting a reliable alternative diagnostic tool for on-site/bed-site clinical analysis.

SPR enhanced DNA biosensor for sensitive detection of donkey meat adulteration.

Herein, a surface plasmon resonance (SPR) enhanced DNA biosensor has been developed for real-time detection of donkey meat marker using biotinylated reporter and streptavidin functionalized gold nanostars (Stre@GNSs). Compared to the direct detection assay, this sandwich format for the enhancement of the signal, resulted in 6-folds orders increase in the sensitivity. Target DNA could be detected with the lower limit of quantification (LLOQ) of 1.0nM with a relative standard deviation (RSD, n=3) of 0.85%. In addition, the fabricated SPR sensor showed good selectivity for the target analyte over full complementary, single-base mismatch, three base-mismatch and non-complementary oligonucleotides. Finally, the proposed sensor was successfully applied for detection of donkey meat adulteration with various percentages in homemade beef sausage, as a real sample. The results indicated that the proposed biosensor provides a high specificity, easy, good sensitivity and fast approach for identification of donkey meat adulteration in food samples.

A strip test for the optical determination of influenza virus H3 subtype using gold nanoparticle coated polystyrene latex microspheres.

A strip test is described for the optical determination of influenza virus H3 subtype. It utilizes gold nanoparticle (AuNP) coated polystyrene latex microspheres (PS) as the label and a sandwich format. The AuNP and PS particles were linked using monoclonal antibodies against influenza virus as the bridge. Under the optimal conditions, the visual detection limit of the AuNP-PS-based strip test was as low as 1/16 hemagglutination unit (HAU). It was 64 times higher than that of 10nm (4 HAU) AuNP-based strip tests. Quantitative analysis showed that the detection limit of the AuNP-PS-based strip is 0.016 HAU. The AuNP-PS-based strip test showed no cross-reactivity to the other subtypes (H1, H5, H7, or H9) of influenza viruses. Graphical abstract .

Antibody-Electroactive Probe Conjugates Based Electrochemical Immunosensors.

Suitable immobilization of a biorecognition element, such as an antigen or antibody, on a transducer surface is essential for development of sensitive and analytically reliable immunosensors. In this review, we report on (1) methods of antibody prefunctionalization using electroactive probes, (2) methods for immobilization of such conjugates on the surfaces of electrodes in electrochemical immunosensor construction and (3) the use of antibody-electroactive probe conjugates as bioreceptors and sensor signal generators. We focus on different strategies of antibody functionalization using the redox active probes ferrocene (Fc), anthraquinone (AQ), thionine (Thi), cobalt(III) bipyridine (Co(bpy)33+), Ru(bpy)32+ and horseradish peroxidase (HRP). In addition, new possibilities for antibody functionalization based on bioconjugation techniques are presented. We discuss strategies of specific, quantitative antigen detection based on (i) a sandwich format and (ii) a direct signal generation scheme. Further, the integration of different nanomaterials in the construction of these immunosensors is presented. Lastly, we report the use of a redox probe strategy in multiplexed analyte detection.

Ultrasensitive and Selective Determination of Carcinoembryonic Antigen Using Multifunctional Ultrathin Amino-Functionalized Ti3C2-MXene Nanosheets

Herein, we report on a two-dimensional amino-functionalized Ti3C2-MXene (N-Ti3C2-MXene)-based surface plasmon resonance (SPR) biosensor for detecting carcinoembryonic antigen (CEA) utilizing a sandwich format signal amplification strategy. Our biosensor employs an N-Ti3C2-MXene nanosheet-modified sensing platform and a signal enhancer comprising N-Ti3C2-MXene-hollow gold nanoparticles (HGNPs)-staphylococcal protein A (SPA) complexes. Ultrathin Ti3C2-MXene nanosheets were synthesized and functionalized with aminosilane to provide a hydrophilic-biocompatible nanoplatform for covalent immobilization of the monoclonal anti-CEA capture antibody (Ab1). The N-Ti3C2-MXene/HGNPs nanohybrids were synthesized and further decorated with SPA to immobilize the polyclonal anti-CEA detection antibody (Ab2) and serve as signal enhancers. The capture of CEA followed by the formation of the Ab2-conjugated SPA/HGNPs/N-Ti3C2-MXene sandwiched nanocomplex on the SPR chip results in the generation of a response signal. The fabricated N-Ti3C2-MXene-based SPR biosensor exhibited a linear detection range of 0.001-1000 PM with a detection limit of 0.15 fM. The proposed biosensor showed high sensitivity and specificity for CEA in serum samples, which gives it application potential in the early diagnosis and monitoring of cancer. We believe that this work also opens new avenues for development of MXene-based highly sensitive biosensors for determining various biomolecules.

Electrochemical genosensor for Klotho detection based on aliphatic and aromatic thiols self-assembled monolayers

Changes in expression of Klotho gene are associated with chronic kidney disease and its potential as early biomarker is being studied. We report, for the first time, the detection of Klotho gene by a biosensor platform. Self-assembled mixed monolayers (SAMs) as DNA immobilization method in screen-printed gold electrodes and a sandwich format detection were used in the development of an electrochemical genosensor for the detection of a 100-mer DNA fragment, copy of the partial region of the mRNA Klotho gene. The use of different binary and ternary SAMs based on aliphatic (mercaptohexanol, MCH, and hexanedithiol, HDT) and aromatic (mercaptophenylacetic acid, MPAA) thiol diluents and capture probe (CP) as sensing phases was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. Multiple configurations were studied, changing the order of component addition and comparing co-immobilization and two-step immobilization processes. The procedure for binary SAM preparation consisting of sequential addition of a thiol diluent followed by CP was found to have the least detrimental impact on electrochemical performance. The signal-to-blank ratios increased considerably in the case of thioaromatic binary DNA monolayers, MPPA/CP, compared to the values obtained for aliphatic SAMs. Ternary monolayers formed by MCH and HDT rendered good fractional coverage levels and generated more reversible redox reactions at the surface, mostly when CP was firstly immobilized, CP/HDT/MCH. A significant reduction of the blank and non-specific (non-complementary sequence) signals was obtained with this ternary SAM, compared to binary SAMs and an increase of 2.42-fold of the S/B ratio (10 nM of target) compared with MPAA/CP SAMs. A linear response in the range of 5·10−10 to 5·10−8 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.

Self-assembled colloidal gold superparticles to enhance the sensitivity of lateral flow immunoassays with sandwich format.

Background: Traditional lateral flow immunoassay (LFIA) based on 20-40 nm gold nanoparticles (AuNPs) as signal reporter always suffers from relatively low detection sensitivity due to its insufficient brightness, severely restricting its wide-ranging application in the detection of target analytes with trace concentration.Methods: To address this problem, the self-assembled colloidal gold superparticles (GSPs) were synthesized as an improved absorption-dominated labeling probe for improving the sensitivity of sandwich LFIA. Five kinds of GSPs with the size ranging from 100 nm to 400 nm were synthesized by embedding hydrophobic AuNPs of size 12 nm as building blocks into the polymer nanobeads. The as-prepared GSPs were suggested as novel labeling probes of LFIA. The effects of the size of assembled GSPs on the sensitivity of sandwich LFIA was assessed, and the detection performance of GSPs-LFIA was further compared with traditional AuNPs-LFIA.Results: The resultant GSPs showed extremely high light absorption but very low light scattering, which favor the absorption-dominated signal output in LFIA. Among them, the GSP270-LFIA (size 270 nm) exhibits the highest sensitivity for human chorionic gonadotropin and hepatitis B surface antigen detection in real serum sample, which are approximate 39.79- and 13.8-fold higher than that of traditional AuNP40-LFIA.Conclusions: The proposed research demonstrated that the current GSPs can provide an ultrasensitive and quantitative detection for disease biomarkers in real serum samples as promising reporters of sandwich LFIA platform.

First Report of Natural Infection of Siraitia grosvenorii by Cucumber Green Mottle Mosaic Virus in China

Fruit of Siraitia grosvenorii (family Cucurbitaceae) are used as a sweetener in beverage and traditional herbal medicine production in China. Some viruses have been known to infect S. grosvenorii, such as papaya ringspot virus (PRSV), watermelon mosaic virus (WMV), and zucchini yellow mosaic virus (ZYMV) (Cai et al. 2001; Liao et al. 2005; Qin et al. 2005). In August 2017, six S. grosvenorii samples (nos. LHG-1 to LHG-6) that showed mosaic, chlorosis, and mottling of leaves were collected from Yongfu, Guangxi province, China. All symptomatic samples were tested by enzyme-linked immunosorbent assay (ELISA) in double antibody sandwich format for 10 known cucurbit-infecting viruses, including cucumber green mottle mosaic virus (CGMMV), cucumber mosaic virus, kyuri green mottle mosaic virus, PRSV, squash mosaic virus, tobacco mosaic virus (TMV), watermelon silver mottle virus, WMV, zucchini green mottle mosaic virus, and ZYMV. The sample LHG-1 with chlorosis and mottle symptoms reacted positively against the antisera of CGMMV and TMV (Adgen, U.K.) but negatively against antisera of the other viruses. The other samples were negative in ELISA for all viruses tested. Total RNA was extracted from the LHG-1 sample using TRIzol reagent (Takara Dalian, China) following the manufacturer’s instructions. Reverse transcription polymerase chain reaction (RT-PCR) assays using CGMMV-specific primer pair CGM1/CGM2 (Huang et al. 2007) and TMV-specific primers (Li et al. 2000) showed a positive RT-PCR product of the expected size (650 bp) for CGMMV but not for TMV. The amplicon was further cloned into pMD-18-T vector (Takara Dalian) and sequenced (GenBank accession no. MK858182). Sequence analysis revealed that the RT-PCR product showed 99% nucleotide identity to the sequences of CGMMV isolates available in GenBank (accession nos. HM008919, KY753928, KX883833, KP868654, KM873786, and MH271443). The virus in LHG-1 was therefore identified as an isolate of CGMMV and named as CGMMV-LHG. Additionally, to confirm the infectivity of CGMMV-LHG, leaf sap extract in phosphate buffer (0.1 M, pH 7.0) of LHG-1 was mechanically inoculated on leaves of S. grosvenorii and Lagenaria siceraria seedlings. Ten days after inoculation, S. grosvenorii produced symptoms similar to the diseased plants in the field, and systemic mosaic and vein-banding were observed on L. siceraria. The control seedlings inoculated with phosphate buffer remained symptomless. Presence of CGMMV in inoculated plants was confirmed by RT-PCR. To the best of our knowledge, this is the first report of CGMMV infection on S. grosvenorii under natural conditions. The virus can be transmitted by seeds, infects various cucurbit species, and causes yield losses all over the world (Liu et al. 2014; Wu et al. 2011). Use of virus-free seeds and seedlings will be helpful to control CGMMV in S. grosvenorii production areas.

Ultrasensitive aptamer-based protein assays based on one-dimensional core-shell nanozymes

In enzyme-based immunoassys, the use of natural enzyme has been remarkably restricted by the inconvenience in preparation and storage, especially for point-of-care testing. Nanozymes, which can mimic the functions of natural enzymes, have been regarded as promising alternatives due to their robust stability and convenience in fabrication. Here we fabricated one-dimensional Fe3O4@C core-shell nanostructures via a solvent-thermal method. Thus prepared nanocomposites showed excellent peroxidase-like activity, capable of catalyzing chromogenic substrates into colored products in the presence of H2O2. We then developed a nanozyme-linked aptamer sorbent assay (NLASA) in a sandwich format, in which the as-prepared Fe3O4@C nanowires were employed as catalytic labels for colorimetric detection by naked eyes. In the detection of platelet-derived growth factor BB (PDGF-BB), this assay reliably exhibited detection limits as low as 10 fM, with a working range from 10 fM to 100 nM. By incorporating G-quadruplex-hemin DNAzyme with Fe3O4@C nanowires, the detection limit could be further lowered to 50 aM. The detection limit of PDGF-BB in 50% human serum was 100 fM. This ultrasensitive, cost-effective and easy-to-operate sensing platform offers new opportunities for protein detection in clinical diagnosis.

Design and Simple Assembly of Gold Nanostar Bioconjugates for Surface-Enhanced Raman Spectroscopy Immunoassays.

Immunoassays using Surface-Enhanced Raman Spectroscopy are especially interesting on account not only of their increased sensitivity, but also due to its easy translation to point-of-care formats. The bases for these assays are bioconjugates of polyclonal antibodies and anisotropic gold nanoparticles functionalized with a Raman reporter. These bioconjugates, once loaded with the antigen analyte, can react on a sandwich format with the same antibodies immobilized on a surface. This surface can then be used for detection, on a microfluidics or immunochromatographic platform. Here, we have assembled bioconjugates of gold nanostars functionalized with 4-mercaptobenzoic acid, and anti-horseradish peroxidase antibodies. The assembly was by simple incubation, and agarose gel electrophoresis determined a high gold nanostar to antibody binding constant. The functionality of the bioconjugates is easy to determine since the respective antigen presents peroxidase enzymatic activity. Furthermore, the chosen antibody is a generic immunoglobulin G (IgG) antibody, opening the application of these principles to other antibody-antigen systems. Surface-Enhanced Raman Spectroscopy analysis of these bioconjugates indicated antigen detection down to 50 µU of peroxidase activity. All steps of conjugation were fully characterized by ultraviolet-visible spectroscopy, dynamic light scattering, -Potential, scanning electron microscopy, and agarose gel electrophoresis. Based on the latter technique, a proof-of-concept was established for the proposed immunoassay.

Impact of assay format on miRNA sensing: Electrochemical microfluidic biosensor for miRNA-197 detection

MicroRNAs (miRNAs) are important biomarkers for the early detection of various diseases, especially cancer. Therefore, there is a continuing interest in different biosensing strategies that allow for the point-of-care measurement of miRNAs. Almost all miRNA sensors utilize cross-hybridization of the target miRNA with a capture probe for the recognition, which can be designed in either a sandwich or a competitive format. In this work, we present a low-cost microfluidic biosensor platform for the electrochemical measurement of miRNA-197 (a tumor biomarker candidate) in undiluted human serum samples, operating with very low sample volumes (580 nl) and a sample-to-result time of one hour. For this purpose, different on-chip miRNA bioassays based on sandwich and competitive formats are developed and compared in terms of their sensitivity, dynamic range, selectivity, precision, and simplicity. The obtained results show that, despite having a narrower dynamic range when compared to the competitive format, the sandwich assay has superior performance regarding its sensitivity and selectivity. The lowest limit of detection which can be achieved with the sandwich assay is 1.28 nM (0.74 fmole), while 4.05 nM (2.35 fmole) with the competitive format. Moreover, the sandwich assay proves to have a better distinction against single-base mismatch oligonucleotide sequences compared to the competitive one. Due to its versatility and easy handling, overcoming the issue with the sensitivity, the implemented electrochemical microfluidic biosensor could pave the way for rapid and low-cost on-site miRNA diagnostics.