Lijuan Bai

Simultaneous electrochemical detection of multiple analytes based on dual signal amplification of single-walled carbon nanotubes and multi-labeled graphene sheets

In this work, a sandwich-type electrochemical aptasensor for simultaneous sensitive detection of platelet-derived growth factor (PDGF) and thrombin is fabricated. Reduced graphene oxide sheets (rGS) are used as matrices to immobilize the redox probes, which are subsequently coated with platinum nanoparticles (PtNPs) to form the PtNPs-redox probes-rGS nanocomposites. With the employment of the as prepared nanocomposites, a signal amplification strategy was described based on bienzyme (glucose oxidase and horseradish peroxidase) modified PtNPs-redox probes-rGS nanocomposites as the tracer labels for secondary aptamers (Apt II) through sandwiched assay. Gold nanoparticles functionalized single-walled carbon nanotubes (AuNPs@SWCNTs) as the biosensor platform enhance the surface area to capture a large amount of primary aptamers (Apt I), thus amplifying the detection response. The experiment results show that the multi-labeled PtNPs-redox probes-rGS nanocomposites display satisfying electrochemical redox activity and highly electrocatalytic activity of PtNPs and bienzyme, which exhibit high sensitivity for detection of proteins. The linear range of PDGF is 0.01-35 nM with a detection limit of 8 pM, while the linear ranges from 0.02 to 45 nM and a detection limit of 11 pM for thrombin are obtained.

Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection.

In this work, we reported a sandwiched luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant. In order to construct the base of the immunosensor, a hybrid architecture of Au nanoparticles and graphene by reduction of HAuCl(4) and graphene oxide (GO) with ascorbic acid was prepared. The resulted hybrid architecture modified electrode provided an excellent platform for immobilization of antibody with good bioactivity and stability. Then, ZnONPs and GOD functionalized graphene labeled secondary antibody was designed for fabricating a novel sandwiched ECL immunosensor. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol-H(2)O(2) system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of carcinoembryonic antigen (CEA) in the range from 10 pg mL(-1) to 80 ng mL(-1) and with a detection limit of 3.3 pg mL(-1) (SN(-1)=3). The amplification strategy performed good promise for clinical application of screening of cancer biomarkers.

Development of an electrochemical method for Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is an outstanding DNA amplification procedure, in which the reaction can accumulate 10(9) copies from less than 10 copies of input template within an hour. While the amplification reaction is extremely powerful, the quantitative detection of LAMP products is still analytically difficult. Besides, the type of targets that LAMP can detect is also less, which to some extent limited the application of LAMP. In this study, we are reporting for the first time an efficient and accurate detection system which employs the integration of LAMP, aptamer and the electrochemical method for the sensitive detection of Ochratoxin A (OTA). Aptamers were designed as the forward outer primer to trigger the LAMP reaction, and then the LAMP amplification products were combined with a redox active molecule methylene blue (MB) and analyzed by an electrode using differential pulse voltammograms (DPV). As the reaction progresses, the MB intercalated into double-stranded regions of LAMP amplicons reduces the free MB concentration. Hence, the peak current of reaction mixture decreased with the amplification because of the slow diffusion of MB-amplified DNA complex to the electrode surface. The peak height of the current was related to the input amount of the aptamers, providing a ready means to detection the concentration of OTA. With such design, the proposed assay showed a good linear relationship within the range of 0.001-50 nM with a detection limit of 0.3 pM (defined as S/N = 3) for OTA.

Dendrimer functionalized reduced graphene oxide as nanocarrier for sensitive pseudobienzyme electrochemicalaptasensor

A novel sensitive sandwich-type pseudobienzyme aptasensor was developed by dendrimer functionalized reduced graphene oxide (PAMMA-rGO) as nanocarrier and hemin/G-quadruplex as NADH oxidase and HRP-mimicking DNAzyme. Greatly enhanced sensitivity for the target thrombin was achieved by using a dual signal amplification strategy: first, the PAMMA-rGO not only constructed an effective platform for anchoring larger amounts of electron mediator thionine (TH) and hemin/G-quadruplex bioelectrocatalytic complex with high stability and bioactivity but also accelerated the electron transfer process assisted by the conductive rGO with amplified electrochemical signal output. Second, the hemin/G-quadruplex simultaneously acting as an NADH oxidase and HRP-mimicking DNAzyme for constructing pseudobienzyme amplifying system could in situ biocatalyze formation of H₂O₂ with high local concentrations and low transfer loss that lead to obvious signal enhancements. On the basis of the dual signal amplification strategy of PAMMA-rGO and the pseudobienzyme amplifying, the developed aptasensor could respond to 0.1 pM thrombin with a linear calibration range from 0.0002 to 30.0 nM. Compared with protein enzymes assisted bienzyme aptasensor, this new aptasensor avoided the fussy labeling process and the spatial distribution of each sequentially acting enzyme, which provided ideal candidate for the development of sensitive and simple bioanalytical platform.

A multi-amplification aptasensor for highly sensitive detection of thrombin based on high-quality hollow CoPt nanoparticles decorated graphene

In this work, we have successfully demonstrated a facile strategy to incorporate high-quality hollow CoPt bimetal alloy nanoparticles (HCoPt) onto reduced graphene oxide sheet (HCoPt-RGs). An advanced sandwich-type electrochemical aptasensor for thrombin was proposed by using the HCoPt-RGs conjugates as secondary label. The formed conjugates provided large surface area for loading plentiful redox probe thionine (Thi), horseradish peroxidase (HRP) and secondary aptamer (Apt II) with good stability and friendly biocompatibility, indicating their superior properties in electroactive mediator enrichment and biomolecule immobilization. Furthermore, activated by glutaraldehyde (GA), the chitosan-hollow CoPt alloy nanoparticle (CS-HCoPt) film can greatly facilitate the capture of primary aptamer (Apt I) and dramatically reduce the nonspecific binding. Excellent sensitivity was obtained by detecting the conspicuously enhanced electrochemical signal of Thi, which was amplified by HCoPt alloy nanoparticles and HRP toward the catalytic reduction of H(2)O(2). The aptasensor displayed excellent performance for thrombin with a wide linearity in the range from 1.0×10(-12) to 5.0×10(-8) M and a relatively low detection limit of 3.4×10(-13) M. Moreover, the resulted aptasensor also exhibited good specificity, acceptable reproducibility and stability, indicating that the present strategy could pave a promising way for the wide application of graphene in clinical research.

Bi-enzyme functionlized hollow PtCo nanochains as labels for an electrochemical aptasensor.

In this work, a new signal amplification strategy based on hollow PtCo nanochains (HPtCoNCs) functionalized by bi-enzyme-horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) and glucose oxidase (GOD), as well as ferrocene-labeled secondary thrombin aptamer (Fc-TBA 2), is developed to construct a highly sensitive electrochemical aptasensor. The HRP-DNAzyme contains a special G-quadruplex structure with an intercalated hemin. With the surface area enlarged by HPtCoNCs, the amount of immobilized Fc-TBA 2, hemin and GOD can be enhanced. Under the enzyme catalysis of GOD, d-glucose is rapidly oxidized into gluconic acid accompanying with the generation of H₂O₂, which is further electrocatalyzed by Pt nanoparticles and HPR-DNAzyme to improve the electrochemical signal of Fc. With several amplification factors mentioned above, a wide linear ranged from 0.001 to 30 nM is acquired with a relatively low detection limit of 0.39 pM for thrombin. The present work demonstrates that using HPtCoNCs as labels is a promising way to amplify the analysis signal and improve the sensitivity of aptasensors.

Synthesis of multi-fullerenes encapsulated palladium nanocage, and its application in electrochemiluminescence immunosensors for the detection of Streptococcus suis Serotype 2.

A novel functionalized material is synthesized using surface-decorated fullerene (C60) to encapsulate hollow and porous palladium nanocages (PdNCs), and is applied to fabricate an electrochemiluminescence (ECL) immunosensor for the detection of Streptococcus suis Serotype 2 (SS2). PdNCs with hollow interiors and porous walls are prepared using a galvanic replacement reaction between silver nanocubes and metal precursor salts. Then, C60 reacts with L-cysteine (L-Cys) to form L-Cys functionalized C60 (C60-L-Cys), which has a better biocompatibility, conductivity, and hydrophilicity compared to C60 and possesses abundant -SH groups on the surface. Because of the special interaction between -SH and PdNCs, the obtained C60-L-Cys is adsorbed around the PdNCs to form an interesting structure with multiple spheres encapsulating the cage. The resultant functionalized material (C60 -L-Cys-PdNCs) has a high specific surface area, good electrocatalytic ability, and efficient photocatalytic activity, and is used to construct an ECL immunosensor for the detection of SS2. The ECL signal amplified strategy is performed by using the novel coreactant (C60-L-Cys) and in situ generation of O2 thus creating the S2O8(2-)-O2 ECL system. As a result, a wide linear detection range of 0.1 pg mL(-1) to 100 ng mL(-1) is acquired with a relatively low detection limit of 33.3 fg mL(-1).

Ultrasensitive thrombin detection based on direct electrochemistry of highly loaded hemoglobin spheres-encapsulated platinum nanoparticles as labels and electrocatalysts.

For the first time, a sandwich-type electrochemical method was proposed for ultrasensitive thrombin (TB) detection based on direct electrochemistry of highly loaded hemoglobin spheres-encapsulated platinum nanoparticles (PtNPs@Hb) as labels and electrocatalysts. The prepared PtNPs@Hb not only exhibited good biocompatibility, excellent electrocatalytic activity, but also presented redox activity of Hb. Thus, it was employed for the fabrication of aptasensor without any extraneous redox mediators, leading to a simple preparation process for the aptasensor. The high loading of Hb spheres as redox mediators could enhance the electrochemical signal. Importantly, the synergetic electrocatalytic behavior of Hb and PtNPs toward H2O2 reduction greatly amplified the electrochemical signal, resulting in the high sensitivity of aptasensor. Consequently, under optimal conditions, the designed aptasensor exhibited a lower detection limit of 0.05 pM and wide dynamic linear range from 0.15 pM to 40 nM for TB detection. Additionally, the proposed mediator-free and signal-amplified electrochemical aptasensor showed great potential in portable and cost-effective TB sensing devices.

Direct electrochemistry and electrocatalysis of a glucose oxidase-functionalized bioconjugate as a trace label for ultrasensitive detection of thrombin.

For the first time, a glucose oxidase-functionalized bioconjugate was prepared and served as a new trace label through its direct electrochemistry and electrocatalysis in a sandwich-type electrochemical aptasensor for ultrasensitive detection of thrombin.

Label-free electrochemical aptasensor for sensitive thrombin detection using layer-by-layer self-assembled multilayers with toluidine blue-graphene composites and gold nanoparticles.

In the present study, toluidine blue-graphene (Tb-Gra) nanocomposites were prepared to design a Lable-free electrochemical aptasensor for highly sensitive detection of thrombin based on layer-by-layer (LBL) technology. The nanocomposites with excellent redox electrochemical activities were first immobilized on the gold nanoparticles (nano-Au) modified glassy carbon electrodes (GCE). Then, the LBL structure was performed by electrostatic adsorption between the positively charged Tb-Gra and negatively charged nano-Au, which formed {Tb-Gra/nano-Au}(n) multilayer films for electroactive species enrichment and biomolecule immobilization. Subsequently, the thiolated thrombin binding aptamer (TBA) was assembled on the nano-Au surface through Au-S bond. In the presence of target thrombin (TB), the TBA on the multilayer could catch the thrombin onto the electrode surface, which resulted in a barrier for electro-transfer, leading to the decrease of the electrochemical signal of Tb-Gra nanocomposites. Under the optimal conditions, a wide detection range from 0.001 nM to 80 nM and a low detection limit of 0.33 pM (defined as S/N=3) for thrombin were obtained. In addition, the sensor exhibited excellent selectivity against other proteins.