Han Xia

Development of a Quartz Crystal Microbalance Biosensor with Aptamers as Bio-recognition Element

The ultimate goal in any biosensor development project is its use for actual sample detection. Recently, there has been an interest in biosensors with aptamers as bio-recognition elements, but reported examples all deal with standards, not human serum. In order to verify the differences of aptamer-based biosensor and antibody-based biosensor in clinical detection, a comparison of the performance of aptamer-based and antibody-based quartz crystal microbalance (QCM) biosensors for the detection of immunoglobulin E (IgE) in human serum was carried out. Aptamers (or antibodies) specific to IgE were immobilized on the gold surface of a quartz crystal. The frequency shifts of the QCM were measured. The linear range with the antibody (10–240 μg/L) compared to that of the aptamer (2.5–200 μg/L), but a lower detection limit could be observed in the aptamer-based biosensor. The reproducibility of the two biosensors was comparable. The aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity. In addition, the aptamer receptors could tolerate repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. When stored for three weeks, the frequency shifts of the aptamer-coated crystals were all greater than 90% of those on the response at the first day.

Isothermal detection of multiple point mutations by a surface plasmon resonance biosensor with Au nanoparticles enhanced surface-anchored rolling circle amplification

In this study, we developed a surface plasmon resonance (SPR) DNA biosensor method using surface-anchored rolling circle amplification (RCA) and Au nanoparticles modified probes (AuNPs) to isothermally detect multiple point mutations associated with drug-resistance in multidrug-resistant Mycobacterium Tuberculosis (MDRTB). A set of probes contains an allele-specific padlock probe (PLP), a capture probe and an AuNPs. The linear PLPs, circularized by ligation upon the recognition of the point mutation on DNA targets, hybridize to the capture probes via the specific tag/anti-tag recognition. Upon recognition each point mutation is identified by locating into the corresponding channel on the chip. Then the immobilized primer (capture probe)-template (circular PLP) complex are amplified isothermally as RCA and further amplified by AuNPs. The RCA products immobilized on the chip surface cause great SPR angle changes consequently. The 5 pM synthetic oligonucleotides and 8.2 pg uL(-1) of genomic DNA from clinical samples can be detected by the method. The positive mutation detection is achieved with a wild-type to mutant ratio of 5000:1. The method was demonstrated by targeting five clinically meaningful mutations in MDRTB. Thirty clinical samples were identified and they were in good agreement with the results from sequencing.

Detection of Staphylococcus epidermidis by a Quartz Crystal Microbalance Nucleic Acid Biosensor Array Using Au Nanoparticle Signal Amplification

Staphylococcus epidermidis is a critical pathogen of nosocomial blood infections, resulting in significant morbidity and mortality. A piezoelectric quartz crystal microbalance (QCM) nucleic acid biosensor array using Au nanoparticle signal amplification was developed to rapidly detect S. epidermidis in clinical samples. The synthesized thiolated probes specific targeting S. epidermidis 16S rRNA gene were immobilized on the surface of QCM nucleic acid biosensor arrays. Hybridization was induced by exposing the immobilized probes to the PCR amplified fragments of S. epidermidis, resulting in a mass change and a consequent frequency shift of the QCM biosensor. To further enhance frequency shift results from above described hybridizations, streptavidin coated Au nanoparticles were conjugated to the PCR amplified fragments. The results showed that the lowest detection limit of current QCM system was 1.3×103 CFU/mL. A linear correlation was found when the concentration of S. epidermidis varied from 1.3×103 to 1.3×107 CFU/mL. In addition, 55 clinical samples were detected with both current QCM biosensor system and conventional clinical microbiological method, and the sensitivity and specificity of current QCM biosensor system were 97.14% and 100%, respectively. In conclusion, the current QCM system is a rapid, low-cost and sensitive method that can be used to identify infection of S. epidermidis in clinical samples.

Real-time monitoring of the strand displacement amplification (SDA) of human cytomegalovirus by a new SDA-piezoelectric DNA sensor system

Nucleic acid amplification has long been used in biosensor technologies, such as DNA sensors, DNA chips and microarrays, due to its advantage of high sensitivity in detecting target DNA. However, dynamic monitoring of nucleic acid amplifications with traditional DNA sensors in real-time is difficult since a constant temperature must be maintained during detection. Thus, the piezoelectric sensor, one type of traditional DNA sensor, is not applicable in real-time monitoring PCR due to the dramatic change in temperature that occurs during reaction. In this study, we introduced strand displacement amplification (SDA), an well-developed nucleic acid amplification technique that can work under conditions of constant temperature, into the development of a novel piezoelectric sensor. Using the new SDA-piezoelectric DNA sensor, we designed a stable system for liquid-phase detection, in which the crystal oscillator plate was fixed by an easily adjustable screw-threaded clamping mechanism and successfully applied the new sensor system to real-time SDA monitoring of human cytomegalovirus (HCMV). This new technique overcomes the shortcomings of traditional DNA sensors in real-time monitoring of nucleic acid amplification. The technique has proved to be a markedly simplified procedure with a number of advantages, such as higher sensitivity, better time efficiency, and the ability of dynamic real-time detection.

Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.

Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.

Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations

The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔC q method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.