Yilin Zhao

A novel non-invasive detection method for the FGFR3 gene mutation in maternal plasma for a fetal achondroplasia diagnosis based on signal amplification by hemin-MOFs/PtNPs.

The small amount of cell-free fetal DNA (cffDNA) can be a useful biomarker for early non-invasive prenatal diagnosis (NIPD) of achondroplasia. In this study, a novel non-invasive electrochemical DNA sensor for ultrasensitive detecting FGFR3 mutation gene, a pathogenic gene of achondroplasia, based on biocatalytic signal materials and the biotin-streptavidin system are presented. Notably encapsulation of hemin in metal-organic frameworks-based materials (hemin-MOFs) and platinum nanoparticles (PtNPs) were used to prepare hemin-MOFs/PtNPs composites via a one-beaker-one-step reduction. We utilized hemin-MOFs/PtNPs for signal amplification because the promising hemin-MOFs/PtNPs nanomaterial has remarkable ability of catalyze H2O2 as well as excellent conductivity. To further amplify the electrochemical signal, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA), gold nanoparticles and streptavidin were selected for modification of the electrode to enhance the conductivity and immobilize more biotin-modified capture probe (Bio-CP) through the high specificity and superior affinity between streptavidin and biotin. The electrochemical signal was primarily derived from the synergistic catalysis of H2O2 by hemin and PtNPs and recorded by Chronoamperometry. Under the optimal conditions, this newly designed biosensor exhibited sensitive detection of FGFR3 from 0.1fM to 1nM with a low detection limit of 0.033fM (S/N=3). We proposed that this ultrasensitive biosensor is useful for the early non-invasive prenatal diagnosis of achondroplasia.

A novel DNA biosensor integrated with Polypyrrole/streptavidin and Au-PAMAM-CP bionanocomposite probes to detect the rs4839469 locus of the vangl1 gene for dysontogenesis prediction

The single nucleotide polymorphism (SNP) of the vangl1 gene is highly correlated with Neural Tube Defects (NTDs), a group of severe congenital malformations. It is hindered by the lack of a quantitative detection method. We first propose the use of a DNA biosensor to detect the missense single nucleotide polymorphism (rs4839469 c.346G>A p.Ala116Thr) of the vangl1 gene in this work. Polypyrrole (PPy) and streptavidin were integrated to modify a gold electrode. We took advantage of the PPys good biocompatibility and excellent conductivity. To further accelerate the electron transfer process at the electrode surface, polyamidoamine dendrimer-encapsulated gold nanoparticles (Au-PAMAM) were used, because Au-PAMAM possess a large number of amino groups to load capture probes (CP). Using the biotin-streptavidin system, the Au-PAMAM-CP bionanocomposite probe, which can detect the target DNA, was conjugated to the electrode surface. Under optimal conditions, the DNA biosensor exhibited a wide linear range of 0.1-100 nM with a low detection limit of 0.033 nM (S/N=3). The results suggest that this approach has the potential to be used in clinical research.

Target triggered cleavage effect of DNAzyme: Relying on Pd-Pt alloys functionalized Fe-MOFs for amplified detection of Pb2+

We designed an amplified detection strategy for the sensitive determination of lead ions (Pb2+) based on a target-triggered nuclear acid cleavage of Pb2+-specific DNAzyme as a selectivity interface combined with Pd-Pt alloys modified Fe-MOFs (Fe-MOFs/PdPt NPs) hybrids acting as the signal tag. Streptavidin modified reduced graphene oxide-tetraethylene pentamine-gold nanoparticles (rGO-TEPA-Au) served as a sensor platform for immobilizing more DNAzyme. In the presence of Pb2+, the substrate DNA strand can be specifically cleaved at the ribonucleotide site by DNAzyme to produce a new single-DNA on the interface. Then, the hairpin DNA with hybrid strand matched by its complement to the single-DNA was employed to modify the Fe-MOFs/PdPt NPs bioconjugates for signal amplification. Fe-MOFs/PdPt NPs catalyze hydrogen peroxide (H2O2) to produce the electrochemical signal which was recorded by chronoamperometry. Benefiting from the Pb2+-dependent DNAzyme, the proposed method can selectively detect Pb2+ in the presence of other metal ions. The newly designed biosensor exhibited a good linear relationship ranging from 0.005 to 1000nmolL-1 with a low detection limit of 2pM (S/N = 3) for Pb2+. This Pb2+-dependent DNAzyme based ultrasensitive biosensor showed high sensitivity and selectivity, providing potential application for Pb2+ detection in naturally contaminated sewage and spiked drinking water samples.

A sensitive sandwich-type immunosensor for the detection of galectin-3 based on N-GNRs-Fe-MOFs@AuNPs nanocomposites and a novel AuPt-methylene blue nanorod

We are presenting an electrochemical immunosensor for the determination of Galectin-3 (Gal-3), a biomarker of heart failure. A glassy carbon electrode (GCE) was modified with a film of a composite made from the N-doped graphene nanoribbons immobilized Fe-based-Metal-organic frameworks deposited with Au nanoparticles (N-GNRs-Fe-MOFs@AuNPs). Primary antibody against Gal-3 (Gal-3-Ab1) was immobilized on the Au nanoparticles on the surface of the modified GCE which then was blocked with bovine serum albumin. Signal amplification is crucial for obtaining low detection limits in biosensors. Here, a relatively simple and ultrasensitive sandwich-type electrochemical immunosensor based on novel signal generation and amplification was developed for the determination of Gal-3. A kind of novel redox-active species, AuPt-Methylene blue (MB) (AuPt-MB) nanocomposites, was synthesized by a one-pot method for the first time. Wherein, MB as a kind of the electron transfer mediators in an amperometric biosensor is responsible for electron production and signal amplification. The rod-like AuPt-MB nanohybrids displayed uniform morphology and good electrochemical activity and can combine with the second antibodies against Gal-3 (Gal-3-Ab2). And the AuPt-MB-Ab2 coupled with the N-GNRs-Fe-MOFs@AuNPs-Ab1 to form the sandwich type format that can greatly enhance the biosensors sensitivity. Under optimal conditions, the designed immunosensor exhibited a linear concentration range from 100fgmL-1 to 50ngmL-1, with a low detection limit of 33.33fgmL-1 (S/N = 3) for Gal-3 in spiked serum. Additionally, the designed immunosensor showed acceptable selectivity, reproducibility and stability. The satisfactory results in analyzing human serum samples indicated potential application promising in monitoring biomarkers.

Rapidly accomplished femtomole soluble CD40 ligand detection in human serum: a “green” homobifunctional agent coupled with reduced graphene oxide-tetraethylene pentamine as platform

The analysis of soluble CD40 ligand (sCD40L), which is present at significant levels in the blood of patients with cardiovascular disease, can reveal the severity of the disease at its early stage. However, the current biomarker detection techniques exhibit poor detection limits. To accomplish this main challenge, herein, we demonstrate an assay based on a novel modified electrochemical immunosensor for the ultrasensitive assay of sCD40L in human serum, which uses β-cyclodextrin (CD) and reduced graphene oxide-tetraethylene (rGO-TEPA) as a platform. rGO-TEPA contains a great number of amino groups and has excellent conductivity, which makes it a promising material for application in electrochemical biosensor development. To further improve the solubility and stability of rGO-TEPA, CD was selected. The CD decorated rGO-TEPA film not only improved the electron transfer but also provided more amino-groups for the immobilization of antibodies. For speeding up the immobilization of antibodies, the amine-modified electrodes were functionalized by a “green” conjugation route using a lower toxicity homobifunctional 1,4-phenylene diisothiocyanate (PDITC) linker. This is the first study that challenges electrochemical immunosensors with CD-rGO-TEPA-PDITC as a platform for the detection of biomarkers. Under optimal conditions, sCD40L could be assayed in the range of 0.25 to 50 pg mL−1 with detection limits of 83.3 fg mL−1 (S/N = 3). We demonstrate excellent specificity and show that the proposed assay accurately detects the protein of interest. The results were in agreement with an enzyme linked immunosorbent assay, suggesting that the electrochemical immunosensor may possess potential towards use in clinical applications of the proposed immunosensor.

Sandwich-type biosensor for the detection of α2,3-sialylated glycans based on fullerene-palladium-platinum alloy and 4-mercaptophenylboronic acid nanoparticle hybrids coupled with Au-methylene blue-MAL signal amplification

α2,3-sialylated glycans (α2,3-sial-Gs) are one of the significant tumour biomarkers for the early diagnosis of cancer. In this work, a neoteric sandwich-type biosensor was developed for detecting α2,3-sial-Gs using 4-mercaptophenylboronic acid (4-MPBA) to construct a novel molecular recognition system by the coordination of a boron atom of 4-MPBA to the amide group of Neu5Ac in the α2,3-sial-Gs structure. Amino-functionalized fullerene coupled with palladium-platinum bimetallic alloy nanocrystals (n-C60-PdPt) was synthesized to modify the surface of a glassy carbon electrode (GCE) because the n-C60 nanomaterial affords a large surface area for the on-site reduction of bimetallic alloy nanoparticles and an excellent capacity for electron transfer. Abundant 4-MPBA were immobilized on the n-C60-PdPt, since the 4-MPBA has the mercapto group can combine with PdPt alloy through strong adsorption. Maackia amurensis lectin (MAL) was covalently immobilized on Au-poly (methylene blue) (Au-PMB) acting as the signal amplification components, which was used to recognize the α2, 3-sial-Gs specifically like a second antibody linked on Au-PMB. The differential pulse voltammetry (DPV) current response of the biosensor in 5mL of PBS (0.1M, pH = 7.4) was recorded, and the proposed sandwich-type biosensor showed a wide linear range of 10 fg mL-1 -100ngmL-1 as well as, a low detection limit of 3fgmL-1 (S/N = 3). Furthermore, the proposed method exhibited good recovery and stability, indicating its potential for use in clinical studies.

Cerium dioxide-doped carboxyl fullerene as novel nanoprobe and catalyst in electrochemical biosensor for amperometric detection of the CYP2C19*2 allele in human serum

The disposition dose of clopidogrel is different in CYP2C19*2 gene carriers and non-carriers. High-dose clopidogrel has been recommended to overcome a low-responsiveness to clopidogrel in patients with the CYP2C19*2 gene. To guide the choice of clopidogrel dosage and catalyse a development in the field of personalized therapy, we developed an ultrasensitive electrochemical biosensor to detect CYP2C19*2 gene. We constructed a novel assay based on cerium dioxide (CeO2)-functionalized carboxyl fullerene (c-C60) supported by Pt nanoparticles (c-C60/CeO2/PtNPs) for signal amplification. Au nanoparticles @ Fe-MIL-88NH2 (AuNPs@Fe-MOFs) were synthesized by one-step method as the support platform to enhance the conductivity and immobilize more biotin-modified capture probe (bio-CP) through the superior affinity and specificity between streptavidin and biotin. c-C60/CeO2/PtNPs were labeled with signal probe to form the signal label. After the sandwich reaction of CYP2C19*2 gene between capture probe and the signal label, a distinguishing electrochemical signal from the catalysis of H2O2 by signal label would be observed. Amperometry was applied to record electrochemical signals. Under optimized conditions, the approach showed a good linear dependence between current and the logarithm of CYP2C19*2 gene concentrations in the range of 1 fM to 50nM with a low detection limit of 0.33fM (S/N = 3). The proposed method showed good specificity to target DNA compared with possible interfering substances. More importantly, the fabricated biosensor achieved accurate quantitative detection of CYP2C19*2 gene in human serum samples demonstrated by excellent correlations with standard DNA sequencing and provided a promising strategy for electrochemical biosensor detection of other gene mutations.

A dual-type responsive electrochemical immunosensor for quantitative detection of PCSK9 based on n-C60-PdPt/N-GNRs and Pt-poly (methylene blue) nanocomposites

In this study, a dual-type responsive electrochemical immunosensor was developed for the quantitative detection of proprotein convertase subtilisin/kexin type 9 (PCSK9), a potential biomarker of cardiovascular disease in serum. N-doped graphene nanoribbons (N-GNRs) with good conductivity were used as the sensing matrix modifying the glassy carbon electrode. Palladium platinum alloy (PdPt) nanoparticles with high catalytic performance toward the reduction of hydrogen peroxide (H2O2) were reduced onto amino-functionalized fullerene (n-C60-PdPt) and significantly amplified the electrochemical signal recorded by the amperometric i-t curve. Furthermore, staphylococcus protein A (SPA) with antibody orientation function was introduced to improve the immunoreaction efficiency. Accordingly, a label-free immunosensor was fabricated based on n-C60-PdPt/N-GNRs for the quick detection of PCSK9. Meanwhile, to realize ultrasensitive detection of PCSK9, Pt-poly (methylene blue) (Pt-PMB) nanocomposites synthesized by a one-pot method for the first time were used as a novel signal label, which exhibited uniform morphology as well as good conductivity and produced an electrochemical signal recorded by differential pulse voltammetry (DPV). Herein, a novel sandwich-type immunosensor was designed using n-C60-PdPt/N-GNRs as the sensing matrix and Pt-PMB as the signal label for sensitive detection of PCSK9. Under optimal conditions, the label-free immunosensor showed a linear range of 10pgmL-1 to 100ngmL -1 with a detection limit of 3.33pgmL-1 (S/N=3), and the sandwich-type immunosensor exhibited a linear range of 100 fg mL-1 to 100ngmL -1 with a detection limit of 0.033pgmL-1 (S/N=3) for PCSK9 detection, indicating its potential application in clinical bioassay analysis.

A new sight for detecting the ADRB1 gene mutation to guide a therapeutic regimen for hypertension based on a CeO2-doped nanoprobe

The β1-adrenergic receptor gene (Entrez Gene:ADRB1), as the target of beta-blockers for hypertension, can directly influence the antihypertensive effect of metoprolol in the Chinese population. This therapeutic effect is often hindered by a lack of evidence-based medical information. To address this challenge, we report a novel assay based on graphene oxide and a CeO2 nanocomposite functionalized by 3-aminopropyltriethoxysilane supported Pt nanoparticles (GO/CeO2/PtNPs) as a signal probe. Due to the large specific surface area and good adsorption properties of the GO/CeO2 nanocomposite, large amounts of PtNPs were immobilized, which amplified the electrochemical signal and improved the sensitivity of the biosensor. To further improvement the sensitivity of the biosensor, Streptavidin (SA) was introduced because it can provide more active sites for the immobilization of the biotinylated capture probe (bio-CP). The electrochemical signal was primarily derived from the catalysis of H2O2 by GO/CeO2/PtNPs. Chronoamperometry was applied to record electrochemical signals, which linearly increased with target DNA. Under optimal conditions, the prepared biosensor had a wide linear range from 1fM to 10nM and a low detection limit of 0.33fM in the detecting of ADRB1 gene. Moreover, the proposed method had good stability and recovery, suggesting its potential for use in clinical research.

Multi-purpose electrochemical biosensor based on a “green” homobifunctional cross-linker coupled with PAMAM dendrimer grafted p-MWCNTs as a platform: application to detect α2,3-sialylated glycans and α2,6-sialylated glycans in human serum

Sialylated glycans are crucial molecular targets for cancer diagnosis and clinical research. α2,3-Sialylated glycans and α2,6-sialylated glycans are the predominant sialic acids found in nature. Different expression of the quantity of glycans can result in development of different disease. However, there are no ideal methods for discriminating α2,3-sialylated glycans and α2,6-sialylated glycans. In this work, a multi-purpose biosensor is fabricated for sensitive detection of α2,3-sialylated glycans and α2,6-sialylated glycans. To improve the sensitivity of the biosensor, p-MWCNTs were integrated with PAMAM, as PAMAM has highly branched and abundant amino groups, providing a large available surface area for linking with other substances. To achieve distinguishable recognition, Maackia amurensis lectin (MAL) and Sambucus nigra agglutinin (SNA) were included. To facilitate the lectin fixation, PDITC, a kind of green homobifunctional cross-linker, was selected. Under optimized detection conditions, the linear range of detection for α2,3-sialylated glycans is 10 fg mL−1 to 50 ng mL−1 with a lower detection limit of 3 fg mL−1, and the linear range of detection for α2,6-sialylated glycans is 10 fg mL−1 to 50 ng mL−1 with a detection limit of 3 fg mL−1. This work not only provides a method for distinguishing detection of α2,3-sialylated glycans and α2,6-sialylated glycans, but also provides a reference for future clinical testing.