Xiaoxi Li

Highly sensitive, label-free colorimetric assay of trypsin using silver nanoparticles

Herein, we report a simple, sensitive label-free colorimetric assay of trypsin based on silver nanoparticles (AgNPs) aggregation. Generally, a specially designed short peptide chain acts as both the stabilizer of AgNPs and the substrate of trypsin. In the presence of trypsin, the negatively charged part of peptides will be hydrolyzed, leaving the positively charged dipeptide capped on the surface of AgNPs. The electrostatic property alteration then leads to the AgNPs aggregation in certain salt condition. The solution color may change correspondingly due to the localized surface plasmon resonance, which can be monitored by naked eye and UV-vis spectrophotometry. This novel AgNPs-based colorimetric method for quantitative determination of trypsin has a linear detection range from 2.5 to 200 ng mL(-1) and a rather low detection limit down to 2 ng mL(-1). The determination of trypsin can also be realized in complex biological fluids by the proposed method, demonstrating its great potential utility in the clinical applications in the future.

Gold nanoparticles and cleavage-based dual signal amplification for ultrasensitive detection of silver ions.

Silver ion (Ag(+)) is a highly toxic heavy metal ion to fungi, viruses, bacteria, and animals. Therefore, Ag(+) monitoring in water or food resources has become extraordinarily important within the scope of human health. Here, we report a gold nanoparticles and enzyme cleavage-based dual signal amplification strategy for ultrasensitive detection of Ag(+) using electrochemical techniques. This sensing platform for Ag(+) has an extremely low detection limit of 470 fM, which also has satisfactory selectivity. Thus, it can be directly used in drinking water and lake water samples. Moreover, the strategy proposed in this work may have potential to be further developed as a generalized platform for the detection of other analytes by designing new DNA sequences for specific recognition.

Electrochemical Strategy for Sensing Protein Phosphorylation

We herein report a novel electrochemical method in this paper to monitor protein phosphorylation and to assay protein kinase activity based on Zr(4+) mediated signal transition and rolling circle amplification (RCA). First, substrate peptide immobilized on a gold electrode can be phosphorylated by protein kinase A. Then, Zr(4+) links phosphorylated peptide and DNA primer probe by interacting with the phosphate groups. After the introduction of the padlock probe and phi29 DNA polymerase, RCA is achieved on the surface of the electrode. As the RCA product, a very long DNA strand, may absorb a large number of electrochemical speices, [Ru(NH(3))(6)](3+), via the electrostatic interaction, localizing them onto the electrode surface, initiated by protein kinase A, a sensitive electrochemical method to assay the enzyme activity is proposed. The detection limit of the method is as low as 0.5 unit/mL, which might promise this method as a good candidate for monitoring phosphorylation in the future.

An amperometric biosensor for the detection of hydrogen peroxide released from human breast cancer cells

The rapid, accurate and sensitive determination of hydrogen peroxide (H(2)O(2)) is of great significance in the physiological, pathological and environmental fields. In this work, we have proposed a highly sensitive and selective amperometric biosensor for the detection of extracellular H(2)O(2) released from human breast cancer cells with the help of a sequence-specific peptide. Since the peptide immobilized on the electrode surface can specifically bind with horseradish peroxidase (HRP) in a favorable orientation, which then well promotes the catalytic activities of the immobilized enzyme toward the reaction of o-phenylenediamine and H(2)O(2), the proposed biosensor can detect H(2)O(2) in a wide linear range from 1.0×10(-7) M to 1.0×10(-4) M with a low detection limit of 3.0×10(-8) M. It can be also directly used to efficiently trace extracellular H(2)O(2) released from human breast cancer cells MCF-7. Furthermore, the reproducibility, stability and selectivity of the biosensor are also quite well compared with the previous report, so our biosensor might be potentially useful in physiological and pathological detection of H(2)O(2) in the future.

An electrochemical alkaline phosphatase biosensor fabricated with two DNA probes coupled with λ exonuclease.

In this work we have developed a novel electrochemical biosensor for the detection of alkaline phosphatase (AP) by the use of two complementary DNA probes (DNA 1 and DNA 2) coupled with λ exonuclease (λ exo). Firstly, the 5-phosphoryl end of DNA 1 is dephosphorylated by AP. Then DNA 1 hybridizes with DNA 2, previously modified on a gold electrode surface. In this double-strand DNA, DNA 2 strand will be promptly cleaved by λ exo with its phosphoryl at the 5 end. After the DNA 2 strand is completely digested, DNA 1 will be released from the double strands and then hybridizes with another DNA 2 strand on the electrode surface, thus the cycle of the release of DNA 1 and the digestion of DNA 2 continues. Since the DNA probes may absorb hexaammineruthenium(III) chloride, the electrochemical species, and the removal of the DNA 2 strand from the electrode surface will result in the decrease of the detected electrochemical signal, which is initially activated by AP, an electrochemical biosensor to assay the activity of AP is proposed in this work. This method may have a linear detection range from 1 to 20 unit/mL with a detection limit of 0.1 unit/mL, and the detection of the enzymatic activity in complex biological fluids can also be realized.

Tetrahedral DNA nanostructure-based microRNA biosensor coupled with catalytic recycling of the analyte.

MicroRNAs are not only important regulators of a wide range of cellular processes but are also identified as promising disease biomarkers. Due to the low contents in serum, microRNAs are always difficult to detect accurately . In this study, an electrochemical biosensor for ultrasensitive detection of microRNA based on tetrahedral DNA nanostructure is developed. Four DNA single strands are engineered to form a tetrahedral nanostructure with a pendant stem-loop and modified on a gold electrode surface, which largely enhances the molecular recognition efficiency. Moreover, taking advantage of strand displacement polymerization, catalytic recycling of microRNA, and silver nanoparticle-based solid-state Ag/AgCl reaction, the proposed biosensor exhibits high sensitivity with the limit of detection down to 0.4 fM. This biosensor shows great clinical value and may have practical utility in early diagnosis and prognosis of certain diseases.

Fabrication of a protease sensor for caspase-3 activity detection based on surface plasmon resonance.

Diagnosis of apoptosis is essential to the early detection of therapy efficiency and the evaluation of disease progression. Caspase-3 is supposed to be closely related to cellular apoptosis. We describe here a label-free surface plasmon resonance (SPR) detection of apoptosis based on caspase-3 activity assay through enzyme digestion. An artificial peptide sequence was designed as a substrate of caspase-3 and immobilized on a gold disk through covalent binding. The 4Lys part at the end of the pentadecyl-peptide was designed to form a unique peptide array through electrostatic repulsion. The immobilization of the peptide on the gold surface was carefully characterized by SPR and atomic force microscopy. The catalytic conditions of caspase-3 were optimized with electrochemical impedance spectroscopy. The detection limit of caspase-3 was found at a concentration of 1 pg mL(-1). The activity of caspase-3 in apoptotic cells could also be measured sensitively by the one-step and intuitional SPR response decrease. The fabricated simple and convenient caspase-3 sensor is proposed for application in clinical analysis.

Colorimetric assay for protein detection based on "nano-pumpkin" induced aggregation of peptide-decorated gold nanoparticles.

Small peptide can be used as an effective biological recognition element and provide an alternative tool for protein detection. However, the development of peptide-based detecting strategy still remains elusive due to the difficulty of signal transduction. Herein, we report a peptide-based colorimetric strategy for the detection of disease biomarker by using vascular endothelial growth factor receptor 1 (Flt-1) as an example. In this strategy, N-terminal aromatic residue-containing peptide modified gold nanoparticles (GNPs) can form bulky aggregate by the introduction of cucurbit[8]uril (CB[8]) that can selectively accommodate two N-terminal aromatic residue of peptides simultaneously regardless of their sequences. However, in the presence of Flt-1, the peptide can specifically bind to the protein molecule and the N-terminal aromatic residue will be occupied, resulting in little aggregation of GNPs. By taking advantage of the highly affinitive peptide and efficiency cross-linking effect of CB[8] to GNPs, colorimetric assay for protein detection can be achieved with a detection limit of 0.2 nM, which is comparable with traditional methods. The feasibility of our method has also been demonstrated in spiked serum sample, indicating potential application in the future.

Peptide-based electrochemical approach for apoptosis evaluation.

This paper reports a strategy to assemble apoptotic cells on a solid surface using a peptide as the recognition element. And a peptide-based electrochemical biosensor to directly evaluate apoptosis is described for the first time. The peptide modified on an electrode is designed to contain the sequence that can recognize externalized phosphatidylserine on apoptotic cells, and can then capture the cells onto the electrode surface. In the electrochemical system, the immobilized cells can not only provide significant steric hindrance for electron transfer, but also shield the positive charges of the peptide that can attract negatively charged electrochemical probes. Therefore, the obtained electrochemical signals drop significantly after the incubation of apoptotic cells, which can be used to reveal the apoptosis level. The experimental results of this approach are well in line with other standard methods. Moreover, this electrochemical method is simple, cost-effective, convenient, sensitive, and holds great potential toward apoptosis evaluation, therapeutic effect assessment and deeper cellular biological studies.

A lipase-based electrochemical biosensor for target DNA

AbstractA lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The other end of the probe DNA is linked, via carboxy groups, to magnetic nanoparticles. The binding of target DNA transforms the hairpin structure of the probe DNA and causes the exposure of ester bonds. This results in the release of electro-active ferrocene after hydrolysis of the ester bonds, and in an observable electrochemical response. The quantity of target DNA in the concentration range between 1u2009×u200910−12xa0mol·L−1 and 1u2009×u200910−8xa0mol·L−1 can be determined by measuring the electrochemical current. The method can detect target DNA with rapid response (30xa0min) and low interference.n FigureA lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The method can detect target DNA with rapid response (30xa0min) and low interference.