Jun-Tao Cao

Aptamer/Au nanoparticles/cobalt sulfide nanosheets biosensor for 17β-estradiol detection using a guanine-rich complementary DNA sequence for signal amplification

We have developed a sensitive sensing platform for 17β-estradiol by combining the aptamer probe and hybridization reaction. In this assay, 2-dimensional cobalt sulfide nanosheet (CoS) was synthesized by a simple hydrothermal method with L-cysteine as sulfur donor. An electrochemical aptamer biosensor was constructed by assembling a thiol group tagged 17β-estradiol aptamer on CoS and gold nanoparticles (AuNPs) modified electrode. Methylene blue was applied as a tracer and a guanine-rich complementary DNA sequence was designed to bind with the unbound 17β-estradiol aptamer for signal amplification. The binding of guanine-rich DNA to the aptamer was inhibited when the aptamer captured 17β-estradiol. Using guanine-rich DNA in the assay greatly amplified the redox signal of methylene blue bound to the detection probe. The CoS/AuNPs film formed on the biosensor surface appeared to be a good conductor for accelerating the electron transfer. The method demonstrated a high sensitivity of detection with the dynamic concentration range spanning from 1.0×10(-9) to 1.0×10(-12) M and a detection limit of 7.0×10(-13) M. Besides, the fabricated biosensor exhibited good selectivity toward 17β-estradiol even when interferents were presented at 100-fold concentrations. Our attempt will extend the application of the CoS nanosheet and this signal amplification assay to biosensing areas.

Determination of levofloxacin and norfloxacin by capillary electrophoresis with electrochemiluminescence detection and applications in human urine

A novel method for the determination of norfloxacin (NOR) and levofloxacin (LVX) was developed by CE separation and electrochemiluminesence detection (ECL). The methods for capillary conditioning and the effect of solvent type were studied. Parameters affecting the CE and ECL were also investigated. Under the optimum conditions, the two analytes were well separated within 9 min. The LODs (S/N = 3) in standard solution are 4.8×10–7 mol/L for NOR and 6.4×10–7 mol/L for LVX, respectively. The precisions of intraday and interday are less than 4.2 and 8.1%, respectively. The LOQs (S/N = 10) in real human urine samples are 1.2×10–6 mol/L for NOR and 1.4×10–6 mol/L for LVX, respectively. The applicability of the proposed method was illustrated in the determination of NOR and LVX in human urine samples and the monitoring of pharmacokinetics for NOR. The recoveries of NOR and LVX at different levels in human urine samples were between 84.3 and 92.3%.

A luminol electrochemiluminescence aptasensor based on glucose oxidase modified gold nanoparticles for measurement of platelet-derived growth factor BB.

A sandwich-type luminol electrochemiluminescence (ECL) aptasensor for highly sensitive and selective detection of platelet-derived growth factor BB (PDGF-BB) is fabricated. For this proposed ECL aptasensor, a multilayered AuNPs-electrochemically reduced graphene (AuNPs-EG) nanocomposite film was formed on the GCE surface as the base of the aptasensor through a co-electrodeposition method. The AuNPs-EG composites possess high conductivity to promote the electron transfer at the electrode interface and good biocompatibility and large surface area to capture large amounts of primary aptamer (Apt1), thus amplifying the detection response. Moreover, glucose oxidase (GOD) functionalized AuNPs labeled secondary aptamer (GOD-Apt2-AuNPs) was designed as the signal probe for the sandwiched aptasensor. Enhanced sensitivity was obtained by in situ generation of H2O2 from reaction between GOD and glucose and the excellent catalytic behavior of AuNPs to the ECL of the luminol-H2O2 system. Under the optimal conditions, the as-prepared ECL aptasensor exhibited excellent analytical property for the detection of PDGF-BB in the range from 1.0×10(-13) to 5.0×10(-10) mol L(-1) with a detection limit of 1.7×10(-14) mol L(-1) (S/N=3). The application of the present protocol was demonstrated by analyzing PDGF-BB in human serum and human urine samples with the recoveries from 85.0% to 110%.

A novel sandwich electrochemiluminescence aptasensor based on molybdenum disulfide nanosheet–graphene composites and Au nanoparticles for signal amplification

In this work, we constructed a novel sandwich electrochemiluminescence (ECL) aptasensor for sensitive detection of thrombin based on molybdenum disulfide nanosheet–graphene (MoS2–GR) composites and Au nanoparticles. In the protocol, MoS2–GR composites were firstly synthesized and assembled on the glassy carbon electrode (GCE) to promote electron transfer. Subsequently, gold nanoparticles (AuNPs) were covered on the electrode to improve the immobilized amount of aptamer1 (Apt1) that could further amplify the ECL signal as well. Afterwards, Apt1 was conjugated to the electrode via Au–S bonds. Finally, the target thrombin and the quantum dot labeled aptamer2 (QD-Apt2) as signal probes were successively attached to the GCE to fabricate a sandwich ECL aptasensor. With the excellent features of the MoS2–GR composites, AuNPs, and sandwich structures, multiple signal amplification for the ECL aptasensor has been achieved. The ECL intensity depended linearly on the logarithm of the thrombin concentration in the range from 0.02 to 5.0 pM with a detection limit of 1.3 fM (S/N = 3). Furthermore, the aptasensor was successfully applied to the determination of thrombin in human plasma with the recoveries of 90.0–102.4% and the RSDs of 2.1–3.7%. The protocol could serve as a new tool for protein detection in biochemical analysis.

Fabrication of electrochemiluminescence aptasensor based on in situ growth of gold nanoparticles on layered molybdenum disulfide for sensitive detection of platelet-derived growth factor-BB

Molybdenum disulfide–gold nanoparticle (MoS2–AuNP) composites were synthesized by the simple method of in situ growth of AuNPs on layered MoS2. The MoS2–AuNP composites were characterized by scanning electron microscopy and transmission electron microscopy. The results showed that the AuNPs were uniformly decorated on the layered MoS2. A sandwich ECL aptasensor was fabricated via layer-by-layer assembly for detection of platelet-derived growth factor BB (PDGF-BB) using MoS2–AuNP composites as a matrix and quantum dots as a signal probe. The MoS2–AuNP composites exhibited good conductivity and enhanced the immobilized amount of aptamer1 and further generated amplified ECL signals. Under the optimal conditions, the ECL aptasensor has high sensitivity, selectivity and stability for PDGF-BB detection with a wider linear range from 0.01 pmol L−1 to 100 pmol L−1 and a lower detection limit of 1.1 fmol L−1 (S/N = 3). Moreover, the aptasensor has been used for the determination of PDGF-BB in human serum samples with recoveries of 88.0–100.1% and RSDs of 1.6–4.3%. This work paves a novel way for functionalization of MoS2 with AuNPs and provides a promising strategy for the development of various ECL aptasensors.

Sensitive detection of tumor marker CA15‐3 in human serum by capillary electrophoretic immunoassay with chemiluminescence detection

A new and sensitive non-competitive immunoassay (IA) for tumor marker carbohydrate antigen 15-3 (CA15-3) by CE coupling with ECL detection has been developed. This method is based on luminol-H(2)O(2 )reaction catalyzed by horseradish peroxidase (HRP). The optimum CE separation and CL detection conditions were investigated. After the non-competitive immunoreaction, the free HRP-labeled CA15-3 antibody (Ab*) and the bound Ab*-antigen (Ab*-Ag) complex were separated in a separation capillary and then catalyzed the CL reaction of luminol and H(2)O(2 )in a reaction capillary following the separation capillary. The calibration curve based on the peak areas of Ab*-Ag complex plotted against the concentrations of CA15-3 is in the range of 0-250 U/mL with a correlation coefficient of 0.9983 and the detection limit is 0.035 U/mL (S/N = 3). The response for five consecutive injections of 125 U/mL CA15-3 resulted in RSDs of 0.83% and 3.1% for the migration time and the peak area, respectively. The method was successfully used for the quantification of CA15-3 in human sera obtained from healthy persons and from patients with breast cancer.

A novel immunosensing platform for highly sensitive prostate specific antigen detection based on dual-quenching of photocurrent from CdSe sensitized TiO2 electrode by gold nanoparticles decorated polydopamine nanospheres.

Herein, a novel photoelectrochemical (PEC) immunosensing platform for highly sensitive detection of prostate specific antigen (PSA) was constructed based on dual-quenching of photocurrent from CdSe sensitized TiO2 electrode by gold nanoparticles decorated dopamine-melanin nanospheres (AuNPs-Dpa-melanin CNSs). In this proposal, CdSe sensitized TiO2 was used as photoelectrochemical matrix and the functional AuNPs-Dpa-melanin CNSs were used as signal quenching element. The dual quenching of the gold nanoparticles decorated Dpa-melanin CNSs to the CdSe sensitized TiO2 was achieved as follows: (i) the strong energy transfer between the CdSe quantum dots (QDs) and Au NPs diminishes the photocurrent signal of CdSe QDs; (ii) the steric hindrance of AuNPs-Dpa-melanin CNSs partly obstructs the diffusion of the electron donor, i.e. ascorbic acid, to the surface of photoelectrode, which make the depleting efficiency of the photogenerated holes decrease, leading to a declined photocurrent intensity. On the basis of the dual quenching effect of AuNPs-Dpa-melanin CNSs, a competitive immunosensing platform for PSA was designed upon the specific binding of anti-PSA to PSA and PSA functionalized AuNPs-Dpa-melanin CNSs conjugates. This proposed immunosensor possesses wide linear range from 1.0×10-11gmL-1 to 1.0×10-7gmL-1 with the detection limit of 2.7pgmL-1. Moreover, the applicability of the present method was demonstrated in the determination of PSA in human serum. The strategy creates new paradigms for PSA and other tumor markers detection and demonstrates high sensitivity, good specificity, and satisfied applicability in complex biological samples.

Chemiluminescence detection of protein in capillary electrophoresis using aptamer-functionalized gold nanoparticles as biosensing platform.

Highly sensitive and selective detection of disease-related proteins play critical roles in clinical practice and diagnostic assays. Herein, we proposed a highly selective and ultrasensitive chemiluminescence (CL) method for protein detection in capillary electrophoresis (CE) using aptamer-functionalized gold nanoparticles (AuNPs) as biosensing platform. In this protocol, AuNPs were synthesized and conjugated with aptamer to form AuNPs-aptamer. Using thrombin and thrombin binding aptamer as an initial proof-of-concept recognization pair, AuNPs-aptamer was linked to thrombin to produce an AuNPs-aptamer-thrombin complex. The resulted complex and unbound AuNPs-aptamer were separated in CE and detected with luminol-H2O2 CL system. The developed strategy produced an ultrasensitive detection of thrombin down to 13.5 fmol/L (S/N=3) with a linear range from 0.033 to 66.0 pmol/L. The application of the present protocol was demonstrated by analyzing thrombin in human plasma samples with the recoveries of 87.6-116.8%. This novel strategy has many outstanding merits including high specificity of aptamer, excellent catalysis behavior of AuNPs, high sensitivity of CL detection, and high separation efficiency of CE.

Label-free and sensitive electrochemiluminescence aptasensor for the determination of 17β-estradiol based on a competitive assay with cDNA amplification

A label-free competitive electrochemiluminescence (ECL) aptasensor for the determination of 17β-estradiol (E2) was developed based on the immobilization of thiol-capped E2 aptamers onto a gold electrode. A complementary DNA (cDNA) was designed to be used as a detection probe to bind with any unbound E2 aptamer. Tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) was used as an ECL indicator and was electrostatically bound to the E2 aptamer; it competes with the cDNA for aptamer binding sites. With less E2, increased amounts of cDNA can bind to the E2 aptamer and adsorb more Ru(bpy)32+, greatly enhancing the ECL intensity. With more E2, the aptamer will capture E2 and bind less cDNA with an accompanying decrease in Ru(bpy)32+ and a decrease in ECL intensity. Thus, a competitive aptasensor based on the specific binding of the E2 aptamer to E2 and cDNA was achieved. The biosensor for E2 possesses a widely linear detection range from 0.01 to 10 nmol L−1 with a detection limit of 1.1 × 10−12 mol L−1 (S/N = 3). The application of the present protocol was demonstrated by determining E2 in human serum, human urine, and tap water samples with recoveries of 89.8–100.0%, 90.0–103.5% and 89.5–95.0%, respectively. The proposed method provides a powerful tool for the rapid and sensitive detection of small molecules in biological and environmental samples.

A rapid, sensitive and label-free sensor for Hg(II) ion detection based on blocking of cysteine-quenching of fluorescent poly(thymine)-templated copper nanoparticles

A simple, rapid, sensitive and label-free fluorescence sensor has been developed for Hg(II) ion detection on the basis of blocking of cysteine-quenching of fluorescent poly(thymine) (poly T)-templated copper nanoparticles (Cu NPs). Fluorescent Cu NPs as fluorescence probe were formed within 5 minutes under mild conditions by using T30 DNA as template. In the presence of cysteine, the fluorescence intensity of T30-templated Cu NPs was found to be quenched effectively, due to the formation of a coordination complex by the Cu–S metal–ligand bond between the Cu NPs and the cysteine. However, when Hg2+ was added, the more stable Hg–S bond was formed through the strong binding preference of cysteine toward Hg2+, causing the cysteine to be far away from the surface of T30-templated fluorescent Cu NPs and hence the fluorescence intensity recovered. Under the optimized conditions, the sensor achieved highly sensitive and selective detection of Hg2+ in the range from 0.5 nM to 100 nM and the detection limit was 0.1 nM. Furthermore, the label-free method was successfully applied in the detection of Hg2+ in lake water samples. Besides, the developed sensor also showed high selectivity, low cost, and simplified operation. Thus, it would hold considerable potential to construct a simple, selective and sensitive fluorescence platform for the detection of Hg(II) ions.