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A highly sensitive and selective electrogenerated chemiluminescence (ECL) biosensor was developed for the determination of matrix metalloproteinase 3 (MMP-3) in serum via the target-induced cleavage of an oligopeptide. One ECL probe (named as Ir-peptide) was synthesized by covalently linking a new cyclometalated iridium(III) complex ([(3-pba)2Ir(bpy-COOH)](PF6)) (3-pba = 3-(2-pyridyl) benzaldehyde, bpy-COOH = 4′-methyl-2,2′-bipyridine-4-carboxylic acid) with an oligopeptide (CGVPLSLTMGKGGK). An ECL biosensor was fabricated by firstly casting Nafion and gold nanoparticles (AuNPs) on a glassy carbon electrode and then self-assembling both of the ECL probes, 6-mercapto-1-hexanol and zwitterionic peptide, on the electrode surface, from which the AuNPs could be used to amplify the ECL signal and Ir-peptide could serve as an ECL probe to detect the MMP-3. Thanks to the MMP-3-induced cleavage of the oligopeptide contributing to the decrease in ECL intensity and the amplification of the ECL signal using AuNPs, the ECL biosensor could selectively and sensitively quantify MMP-3 in the concentration range of 10–150 ng·mL−1 and with both a limit of quantification (26.7 ng·mL−1) and a limit of detection (8.0 ng·mL−1) via one-step recognition. In addition, the developed ECL biosensor showed good performance in the quantization of MMP-3 in serum samples, with a recovery of 92.6% ± 2.8%–105.6% ± 5.0%. An increased level of MMP-3 was found in the serum of rheumatoid arthritis patients compared with that of healthy people. This work provides a sensitive and selective biosensing method for the detection of MMP-3 in human serum, which is promising in the identification of patients with rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

An enzyme immunoassay was developed based on the coulometric measurement of immunoglobulin M (IgM) against Hantaan viruses (HTNV) by using virus-like particles (VLPs) as recognition molecules. The surface functionalization of screen-printed carbon electrodes (SPCEs) was achieved through paste-exfoliated graphene that was modified with a COOH group and a thionine mediator through supramolecular-covalent scaffolds, on SPCEs by using the binder contained in the ink. After the covalent immobilization of the antibody, the sensor was used for the sandwich enzyme immunoassay of IgM against HTNV. By using HTNV VLPs as the second recognization molecules, the resulting sensor efficiently monitored the reaction of IgM against HTNV and anti-IgM antibody with high specificity. By attaching HTNV nucleocapsid protein antibody conjugate with horseradish peroxidase (HRP) onto VLPs, the signal response of the assay was derived from the coulometric measurement of H2O2 reduction mediated by thionine on the electrode surface after the application of a potential (− 0.2 V vs. Ag/AgCl). The ratio of charges measured before or after H2O2 addition was used to quantify IgM because these charges could be used as background charges or total charges, respectively. The ratio exhibited good agreement with IgM concentration within a range 0.1 to 1000 pg mL−1, and a detection limit of 0.06 pg mL−1 was obtained. The assay demonstrated high sensitivity and specificity toward HTNV-specific IgM in serum. [ABSTRACT FROM AUTHOR]

A novel point-of-care testing (POCT) method for the determination of proteases was developed for the first time using a designed disposable capillary-fill device based on the cleavage of electrogenerated chemiluminescence (ECL)-label-tagged peptide probes and enabling elimination of the light-shielding from the magnetic beads (MBs). As a proof-of-principle, prostate-specific antigen (PSA) was taken as a model analyte, and streptavidin-coated magnetic beads bound with ruthenium-complex-tagged specific peptide (biotin-HSSKLQK) were utilized as MB ECL probes. The capillary-fill device was designed to be divided into a reaction zone and detection zone. In the reaction zone, the bio-cleavage reaction between the PSA analyte with the peptide on the surface of the MB ECL probes occurred, while in the detection zone, ECL emission was produced by a screen-printed carbon electrode, Ag/AgCl reference electrode and carbon counter electrode. When the analyte PSA was introduced into the suspension of MB ECL probes in the reaction zone of the device, biocleavage of the peptide occurred, and the cleaved Ru1 part was released from the surface of the MB ECL probes. The capillary-filled device was tilted 90°, and with the aid of gravity, the solution containing the released Ru1 part flowed to the surface of the working electrode in the detection region of the device, while the MB ECL probes were fixed in the reaction zone by an external magnet. PSA can be determined by the ECL emission from the released Ru1 part in the presence of the co-reactant tri-n-propylamine at the detection zone. Under the optimal conditions, the developed ECL method showed a low detection limit of 0.12 ng mL−1 for PSA. This work demonstrates that the developed ECL biosensing approach can eliminate the MB light-shielding effect and quantify proteases with high sensitivity and selectivity, which could be easily extended to POCT-based ECL biosensing for other proteases. [ABSTRACT FROM AUTHOR]

An electrochemiluminescence (ECL) bioassay with high sensitivity and anti-fouling ability was developed for determination of matrix metalloproteinase 9 (MMP-9) secreted from living cells under external stimulation. A peptide with sequence of CLGRMGLPGK and a new cyclometalated iridium(III) complex bearing carboxyl group, (pq)2Ir(dcbpy) (pq = 2-phenylquinoline, dcbpy = 2,2′-bipyridyl-4,4′-dicarboxyli acid, abbreviated as Ir) were employed as molecular recognition substrate and ECL emitter, respectively. The peptide was labelled with the Ir to form Ir-peptide as ECL probe. Ir-peptide was self-assembled onto Nafion and gold nanoparticles (AuNPs) modified glassy carbon electrode (AuNPs/Nafion/GCE) and then both of 6-mercapto-1-hexanol (MCH) and zwitterionic peptide as blocking reagents were co-assembled on Ir-peptide/AuNPs/Nafion/GCE to form an anti-fouling ECL peptide-based biosensor. MMP-9 can be quantified in the range 1.0–50 ng·mL−1 with a detection limit of 0.50 ng·mL−1 based on the decreased ECL intensity. Relative standard derivation was 2.3% for six fabricated anti-fouling ECL peptide-based biosensors after reaction with 50 ng·mL−1 MMP-9. The anti-fouling ECL peptide-based biosensor can be used to monitor MMP-9 secreted from living cells under external stimulation. 96.0%–108.0% of recoveries were obtained in 60-diluted cell culture media. This study demonstrates that the ECL biosensor by the combination of iridium(III) complex-based sensitive ECL method and the anti-fouling interface provides a promising way for the determination of MMP-9 in biological sample, which is viable in clinical diagnosis and point-of-care test of protease. [ABSTRACT FROM AUTHOR]

A homogeneous electrogenerated chemiluminescence (ECL) immunoassay for highly sensitive quantification of specific biomarkers based on immunomagnetic beads and homogeneous detection on a magnetic electrode was developed, for the first time. The magnetic electrode is made of a glassy carbon electrode and a series of ring permanent magnets. D-Dimer antigen was taken as a model analyte while biotinylated D-dimer antibody bound on the streptavidin-coated magnetic beads was utilized as a magnetic capture probe and ruthenium complex-labeled D-dimer antibody was employed as an ECL probe. After a fixed amount of magnetic capture probe and the ECL probe was introduced into analyte D-dimer solution, the "sandwich" immunoconjugates on the magnetic beads were formed in tested solution and then magnetically concentrated on the surface of the magnetic electrode. The homogeneous ECL immunoassay for quantification of specific biomarker was directly carried out in the presence of co-reactant tripropylamine. The low detection limit of 1 ng/mL in magnetic enrich time of 2 min and the good magnetic regeneration for the detection of D-dimer were achieved. The magnetic bead shield ECL emission was extensively discussed. This work demonstrates that the homogeneous (separation-free) ECL immunoassay using magnetic beads and magnetic electrode is a promising approach to quantify the biomarkers with high sensitivity and selectivity and in a short time. This approach can be easily extended to ECL and electrochemical biosensing for other biomarkers. [ABSTRACT FROM AUTHOR]

Abstract Phospholipase A 2 (PLA 2) enzyme could be acted as a unique biomarker for forecasting and diagnosing certain diseases. Therefore, it is important to monitor PLA 2 activity in biological and clinical samples. In this work, a simple electrochemical assay for PLA 2 activity was developed based on a screen-printed carbon electrode (SPCE) with 3D graphene-like surface. When the PLA 2 -containing sample was mixed with the nanoprobes, i.e. the electroactive marker methylene blue (MB) encapsulated within nanometer-sized phospholipid liposomes, MB was released and adsorbed/enriched in site onto the surface of SPCE in a micro-cell. The encapsulation and enzymatic release of MB were evaluated using UV–Vis and fluorescence. The peak current due to oxidation of the adsorbed MB on the SPCE was measured by square-wave voltammetry (SWV). The current was directly linear to the PLA 2 activity from 5 U/L to 200 U/L with a detection limit of 3 U/L. The same method can also be used for screening PLA 2 inhibitors. Thus, the enrichment strategy developed in this work could be a promising signal amplification method for the sensitive and selective detection of PLA 2 in biological or clinical samples. Highlights • A simple electrochemical assay developed for the PLA 2 activity analysis. • 3D graphene-like structure on the SPCE prepared through electrochemical method. • The enzymatic released MB from methylene blue-loaded liposomal nanoprobe adsorbed and enriched on to SPCE. • An approximately 40-fold increase in current obtained on the SPCE with 3D graphene-like surface. [ABSTRACT FROM AUTHOR]

This work reports the performance enhancement strategies on magnetic beads (MBs)-based electrochemiluminescence (ECL) platforms by using double magnetic field actuation of the ECL magnetic microbiosensors (MMbiosensors) for highly sensitive determination of cancer biomarker and exosomes. To obtain the high sensitivity and reproducibility of the ECL MMbiosensors, a series of strategies have been developed including replacing a conventional photomultiplier tube (PMT) with a diamagnetic PMT, replacing the stacked ring-disc magnets with circular-disc magnets lain-in glassy carbon electrode, adding a pre-concentration process of MBs using external magnet actuation. For fundamental research, the ECL MBs taken as the substitute of ECL MMbiosensors were prepared by binding biotinylated DNA tagged with Ru(bpy) 3 2+ derivative (Ru1) to streptavidin-coated MB(MB@SA) were which showed that the developed strategies can enhance 45-fold sensitivity. Importantly, the developed MBs-based ECL platform was estimated by determination of prostate specific antigen (PSA) and exosomes. For PSA, MB@SA•biotin-Ab1(PSA) was taken as the capture probe and Ru1-labeled Ab2 (PSA) was done as ECL probe, while for exosomes, MB@SA•biotin-aptamer (CD63) was taken as the capture probe and Ru1-labeled Ab (CD9) was done as the ECL probe. The experiment results showed that the developed strategies can enhance 33-fold sensitivity of ECL MMbiosensors for PSA and exosomes. The detection limit is 0.28 ng mL−1 for PSA and 4.9 × 102 particle mL-1 for exosomes. This work demonstrated that a series of proposed magnetic field actuation strategies greatly increase the sensitivity of the ECL MMbiosensors. The developed strategies can be expanded to MBs-based ECL and electrochemical biosensors for clinical analysis with greater sensitivity. [Display omitted] • Magnetic field greatly decreases photomultiplier tube response to ECL emission. • Circular-disc magnet contributed uniform distribution of magnetic microbeads. • Magnetic actuation of magnetic microbeads enhances the sensitivity. • High performance of ECL biosensor achieved by magnetic actuation. • Highly sensitive MBs-based ECL biosensor was developed for exosomes. [ABSTRACT FROM AUTHOR]

This work reports washing-free electrogenerated chemiluminescence (ECL) magnetic microbiosensors based on target assistant proximity hybridization (TAPH) for multiple protein biomarkers for the first time. As a principle-of-proof, alpha-fetoprotein (AFP) was chosen as a model analyte, and biotin-DNA1 bound streptavidin-coated magnetic microbeads (MMB@SA⋅biotin-DNA1) were designed as the universal capture MMB, while the corresponding two antibodies tagged with DNA2 or DNA3 were utilized as hybrid recognition probes, and ruthenium complex-tagged DNA4-10A was designed as a universal ECL signal probe. When the capture MMB was added into the mixture solution (containing the analyte, hybrid recognition probes, signal probe and tri- n -propylamine), biocomplexes were formed on the MMB. After the resulting MMB was efficiently brought to the surface of a magnetic glassy carbon electrode (MGCE), ECL measurement was performed without a washing step, resulting in an increase in the ECL intensity. A model for ECL measuring the second-order rate constants of hybridization reactions on MMB was derived. It was found that the rate constants for hybridization reactions on MMB in rotating mode are 1.6-fold higher than those in shaking mode, and a suitable DNA length of the signal probe can improve the signal-to-noise ratio. The washing-free ECL method was developed for the determination of AFP with a much lower detection limit (LOD) of 0.04 ng mL−1. The developed flexible strategy has been extended to determine D-dimer with an LOD of 0.1 ng mL−1 and myoglobinglobin with an LOD of 1.1 ng mL−1. This work demonstrated that the proposed strategy of ECL TAPH on MMB at MGCE is a washing-free and flexible promising strategy, and can be extended to qualify other multiple protein biomarkers in real clinical assays. [Display omitted] • TAPH-based ECL assay on magnetic micro-beads exceed to conventional electrode one. • Highly sensitive ECL methods were developed for α-fetoprotein, D-dimer and myoglobin. • Washing-free ECL magnetic biosensors can be constructed with universal signal probe. • Rotating particles can accelerate biological reaction rates on magnetic beads. • Suitable length of universal signal probe can improve the signal to noise ratio. [ABSTRACT FROM AUTHOR]

A sensitive electrogenerated chemiluminescence (ECL) aptasensor was developed for the detection of human Burkitt’s lymphoma Ramos cells based on the cells-specific oligonucleotide TD05 served as a molecular recognition element and ruthenium(II) complex (Ru1) labeled polyamidoamine dendrimer (Dend) as a signal probe. Dend was used as platform to assemble substantial Ru1 and TD 05 to form Ru1-Dend-SH-(CH 2 ) 6 -oligo(ethylene oxide) 6 -TD05 bioconjugate which was used as the signal probe. Dend was not only used as platform to assemble substantial Ru1 but also used as a coreactant of Ru1 to amplify the ECL signal. The biosensor platform was fabricated by covalently coupling the 52-mer amino-substituted oligonucleotide probes with carboxyl groups on single wall carbon nanotubes-coated glassy carbon electrode. With the sandwich assay, Ramos cells were selectively captured on the surface of the biosensor based on the specific affinity between aptamers and mIgM on the target cells surface, and then, the signal probe was bound with the captured cells. In the presence of tri- n -propylamine, the increased ECL intensity of the biosensor was logarithmically proportion to the concentration of Ramos cells over a range from 1.0 × 10 2 to 1.0 × 10 4 cells/mL with a detection limit of 55 cells/mL. It was found that the sensitivity of the biosensor increased 4.3-fold by comparison with that without Dend. And also, the ECL biosensor showed satisfied selectivity against the tested control cancer cells. [ABSTRACT FROM AUTHOR]

A label-free electrochemical impedance spectroscopy (EIS) biosensor for highly sensitive detection of cardiac troponin I (TnI) as a model target was developed by employing gold nanoparticle (GNPs) modified glassy carbon electrode (GCE) as a base electrode. A special peptide (CFYSHSFHENWPS) was used as molecular recognition probe. GNPs were firstly electrochemically deposited onto the surface of GCE and polyethylene glycol was then used to block any remaining sites of the surface of GCE. The biosensor was fabricated by thiol self-assembling the peptide probe onto the surface of gold nanoparticles and finally blocking any remaining sites of the surface of GNPs using 6-mercapto-1-hexanol. A suitable equivalent electrical circuit consisting of parallel double Randles circuit with phase angle elements greatly fits the experiment electrochemical cell. Upon the binding of TnI, the charge transfer resistance (R ct ) of the biosensor was logarithmically direct proportional to the concentration of TnI in the range from 15.5 pg/mL to 1.55 ng/mL with a detection limit of 3.4 pg/mL. It was found that GNPs modified GCE as a platform for the immobilization of the peptide can greatly reduce the background EIS signal and enhance the EIS response to the target. This biosensor has been used for assay of real serum sample with satisfactory results. This work demonstrates that GNPs modified GCE for the immobilization of molecular recognition element is promising platform for the development of label-free electrochemical biosensors for the determination of special proteins. [ABSTRACT FROM AUTHOR]

A highly selective method for the electrochemical detection of reduced glutathione (GSH) has been developed on the basis of the product of a reaction between GSH and catechol as the electron mediator on single-walled carbon nanotubes modified glassy carbon electrode. A polymerization-adduct ECE mechanism at SWNTs/GCE was proposed. The oxidation peak potential was −0.160 V (vs. Ag/AgCl, sat. KCl) and the oxidation peak current showed a dependence relationship with GSH concentration in the range of 1.0 ∼500.0 μM with a detection limit of 0.5 μM GSH. The developed method can discriminate GSH from other thiol species such as L-cysteine (Cys) and cysteamine hydrochloride (Cym) within their certain concentration ranges. The as-established electrochemical method has been successfully applied for the determination of GSH from the leakage of intracellular GSH contents in HeLa cells following doxorubicin-induced cell apoptosis. [ABSTRACT FROM AUTHOR]

An electrochemical DNA sensor was fabricated based on covalent immobilization of amino-terminated probe DNA onto the polydopamine (PDA) modified screen-printed carbon electrode (SPCE). Immobilization was achieved via one-step Schiff base reaction between the amino group of the probe DNA and quinones in PDA. PDA was formed via electrochemical polymerization of dopamine monomer on the SPCE surface. After a sandwich-type hybridization reaction, the gold nanoparticle-labeled reporter DNA was bound onto the DNA sensor surface to further induce silver deposition in the presence of silver enhancer solutions. The electrochemical stripping signal of the deposited silver nanoparticles in KCl solution was used to monitor the hybridization reaction. Signal amplification enhanced the sensitivity for target DNA detection. The proposed method could detect target DNA at a linear range from 1.0 pM to 70 pM, with a detection limit of 0.3 pM. The DNA sensor exhibited good stability and acceptable reproducibility. The sensor also showed selectivity against non-complementary target DNA. [ABSTRACT FROM AUTHOR]

Highlights: [•] Label-free electrochemical aptasensor was developed for determination of lysozyme. [•] Click chemistry-assisted self-assembly technique was employed as immobilization method. [•] Gold nanoparticles modified screen-printed carbon electrodes were employed as electrode platform. [•] Signal amplification was achieved with detection limit of 0.3pgmL−1 for lysozyme. [ABSTRACT FROM AUTHOR]

Abstract: A label-free electrochemical impedance spectroscopy (EIS) biosensor for the sensitive determination and discrimination of alpha-fetoprotein (AFP) was developed by employing wheat-germ agglutinin (WGA) lectin as molecular recognition element. The EIS biosensor was fabricated by adsorbing carboxyl-functionalized single-wall carbon nanotubes (SWNTs) onto a screen-printed carbon electrode (SPCE) and subsequently covalently coupling WGA onto the surface of the SWNTs-modified electrode. Upon binding of AFP to the biosensor, the electron transfer resistance was increased and the increase in the electron transfer resistance was linearly proportional to the logarithm of the concentration of AFP in the range from 1 to 100ng/L with a detection limit of 0.1ng/L. It was found that the employment of SWNTs as immobilization platform could reduce the background and enhance the EIS response. Moreover, the lectin-based biosensor array fabricated with different lectins was used to evaluate the glycan expression of AFP N-glycan and discriminate AFP between healthy and cancer patients serum samples. This work demonstrates that the employment of carbon nanotubes as immobilization platform and lectin as molecular recognition element in biosensor array is a promising approach for the determination and discrimination of glycoproteins for cancer diagnosis. The strategy proposed in this work could further be used for high-throughput, label-free profiling of the glycan expression of cancer-related glycoproteins and to develop methods for cancer diagnosis in the early stages. [Copyright &y& Elsevier]

Abstract: An ultrasensitive DNA detection method based on coulometric measurement of enzymatic silver deposition on gold nanoparticle (AuNP)-modified screen-printed carbon electrode (SPCE) was developed. The DNA sensor was prepared by self-assembly of a hairpin probe DNA, dually labeled with thiol at its 5′ end and biotin at its 3′ end, on AuNPs on SPCEs. The immobilized probe can shielded the biotin from being approached by a bulky alkaline phosphatase-linked streptavidin (Sv-ALP) conjugate. In the presence of the target DNA, hybridization caused conformational change in the probe DNA and forced biotin away from the surface of the AuNPs. Sv-ALP was bound to the biotin and catalyzed the hydrolysis of ascorbic acid 2-phosphate to form ascorbic acid. The latter reduced the silver ions for the deposition of metallic silver on the electrode surface. The deposited silver was then measured by coulometric analysis in the H2SO4 solution. A considerable increase in signal was obtained because of the accumulation and near-exhaustive coulometric oxidation of metallic silver. Under optimal conditions, the dynamic detection range of the target DNA was from 3.0×10−17 to 1.0×10−14 molL−1, and the detection limit was 1.5×10−17 molL−1. Further, the DNA sensor exhibited selectivity against single-base mismatched DNA. [Copyright &y& Elsevier]

Abstract: A glucose biosensor based on a nanocomposite made by layer-by-layer electrodeposition of the redox polymer into a multilayer containing glucose oxidase (GOx) and single-walled carbon nanotubes (SWCNT) on a screen-printed carbon electrode (SPCE) surface was developed. The objectives of the electrodeposition of redox polymer are to stabilize further the multilayer using a coordinative cross-linked redox polymer and to wire the GOx. The electrochemistry of the layer-by-layer assembly of the GOx/SWCNT/redox polymer nanocomposite was followed by cyclic voltammetry. The resultant biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of layers. The biosensor displayed a linear range from 0.5 to 6.0mM, a sensitivity of 16.4μA/(mMcm2), and a response time of about 5s. It shows no response to 0.05mM of ascorbic acid, 0.32mM of uric acid and 0.20mM of acetaminophen using a Nafion membrane covering the nanocomposite-modified electrode surface. [Copyright &y& Elsevier]

Abstract: An amperometric glucose biosensor based on a multilayer made by layer-by-layer assembly of single-walled carbon nanotubes modified with glucose oxidase (GOx-SWCNT conjugates) and redox polymer (PVI-Os) on a screen-printed carbon electrode (SPCE) surface was developed. The SPCE surface was functionalized with a cationic polymer by electrodeposition of the PVI-Os, followed by alternating immersions in anionic GOx-SWCNT conjugate solutions and cationic PVI-O solutions. The purpose is to build a multilayer structure which is further stabilized through the electrodeposition of PVI-Os on the multilayer film. The electrochemistry of the layer-by-layer assembly of the GOx-SWCNT conjugate/PVI-Os bilayer was followed by cyclic voltammetry. The resultant glucose biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of bilayers. The glucose biosensor displayed a wide linear range from 0.5 to 8.0mM, a high sensitivity of 32μAmM−1 cm−2, and a response time of less than 5s. The glucose biosensor proved to be promising amperometric detectors for the flow injection analysis of glucose. [Copyright &y& Elsevier]