Highly sensitive enumeration of circulating tumor cells in lung cancer patients using a size-based filtration microfluidic chip.

Circulating tumor cells (CTCs) in the peripheral blood could serve as a surrogate marker for the diagnosis of cancer metastasis and for therapeutic evaluation. However, the separation and characterization of CTCs is technically challenging owing to the extremely low number of CTCs present. Here we developed a size-based and high-throughput microfluidic chip, which exploits filtration microchannels to isolate the relatively larger CTCs from the rest of the blood constituents. High isolation efficiency of our microfluidic chip was demonstrated with three lung cancer cell lines spiked in blood samples at an optimal flow rate of 0.4 mL/h. The average recovery rates of 96%, 95% and 92% were obtained for A549, SK-MES-1, and H446, respectively. To clinically validate the chip, we also employed it to isolate CTCs from 59 lung cancer patients. CTCs were detected in 96.7% of patients with the mean number of 18.6 cells/mL, which was significantly higher than normal controls (P<0.05). The work here indicates that the size-based microfluidic platform with the advantage of capturing tumor cells without reliance on cell surface expression markers can provide a novel, inexpensive and effective tool for CTC detection and evaluation of cancer status.

A simple oligonucleotide biochip capable of rapidly detecting known mitochondrial DNA mutations in Chinese patients with Leber's hereditary optic neuropathy (LHON).

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease. Clinically, no efficient assay protocols have been available. In this study, we aimed to develop an oligonucleotide biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing LHON and to investigate frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to LHON were covalently immobilized. Cy5-lablled targets were amplified from blood DNA samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A and 14484 T > C from six confirmed LHON patients were interrogated to validate this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five unrelated healthy individuals were investigated by the LHON biochip, direct sequencing and pyrosequencing, respectively. The biochip was found to be able efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > C or 3460G > A, respectively, accounting for 66.7%. The mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 of 18 cases). The mutations 3460G > A and 3394T > C were found to co-exist in one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller amount of sampling and reaction volume, easier target preparation, fast and high-throughput were the main advantages of the biochip over direct DNA sequencing and pyrosequencing. Our findings suggested that primary mutations of 11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to LHON in China. The biochip would easily be implemented in clinical diagnosis.

Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

Nano-ELISA for highly sensitive protein detection.

Highly sensitive protein detection method based on nanoparticles and enzyme-linked immunosorbent assays (ELISAs), named Nano-ELISA, was introduced. In this method, the micro-magnetic beads were modified with monoclonal antibody of the target protein p53. Gold nanoparticles (AuNPs) were modified with another monoclonal detector antibody and Horseradish peroxidase (HRP, for signal amplification). The presence of target protein p53 causes the formation of the sandwich structures (magnetic beads-target protein-AuNP probes) through the interaction between the antibodies and the antigen p53. The HRP at the surface of AuNPs catalytically oxidize the substrate and generate optical signals that reflected the quantity of the target protein. Down to 5 pg mL(-1) of protein was detected in less than 2 h with this method. The detection sensitivity of p53 classic ELISA kit is 0.125 ng mL(-1). This method is as simple as ELISA and has higher sensitivity than ELISA, which can potentially be exploited in clinic. This method can be used to detect protein markers of tumors, nervous system or other diseases for early diagnostics.

[Development of a DNA biochip for detection of known mtDNA mutations associated with MELAS and MERRF syndromes.].

We developed an oligonucleotide biochip for synchronous multiplex detection of 31 known mitochondrial DNA mutations associated with MELAS (Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MERRF (Myoclonic epilepsy with ragged red fibers). Allele-specific oligonucleotide probes were covalently immobilized on aldehyde modified glass slides, and then hybridized with Cy5-labled DNA fragments amplified from sample DNAs by a multiplex asymmetric PCR (MAP) method. Five patients with MELAS, 5 patients with MERRF and 20 healthy controls were investigated using the oligonucleotide biochip. The results showed that all the cases with MELAS had an A3243G mutation in the MT-TL1 gene. In the MERRF group, 4 cases were found to be an A8344G mutation and 1 case was a T8356C mutation, and both mutations were in the MT-TK gene. In the healthy controls, none of the 31 related mutations was found. The results of the DNA biochip were consistent with those by DNA sequencing. Clearly, the DNA biochip combined with MAP method would become a valuable tool in multiplex detecting of the point mutations in mtDNA leading to MELAS and/or MERRF syndrome. Moreover, this biochip format could be modified to extend to the screening scope of SNPs for any other human mitochondrial diseases.

Microchip-based small, dense low-density lipoproteins assay for coronary heart disease risk assessment.

Small, dense low-density lipoprotein (sdLDL) has been accepted as an emerging cardiovascular risk factor, and there has been an increasing interest in analytical methods for sdLDL profiling for diagnosis. Serum sdLDL may be measured by different laboratory techniques, but all these methods are laborious, time-consuming, and costly. Recently, we have demonstrated that a low-temperature bonding of quartz microfluidic chips for serum lipoproteins analysis (Zhuang, G., Jin, Q., Liu, J., Cong, H. et al., Biomed. Microdevices 2006, 8, 255-261). In contrast to this previous study, we chose SDS as anionic surfactant to modify both lipoproteins and the channel surface to minimize lipoprotein adsorption and improve the resolution of lipoprotein separation. Two major LDL subclass patterns including large, buoyant LDL (lLDL), sdLDL, and high-density lipoprotein (HDL) were effectively separated with high reproducibility. RSD values of the migration time (min) and peak areas of standard LDL and HDL were 6.28, 4.02, 5.02, and 2.5%, respectively. Serum lipoproteins of 15 healthy subjects and 15 patients with coronary heart disease (CHD) were separated by microchip CE. No peaks of sdLDL were detected in serum samples of healthy subjects while sdLDL fractional peaks were observed in patients' entire serum samples. These results suggested that the microchip-based sdLDLs assay was a simple, rapid, and highly efficient technique and significantly improved the analysis of CHD risk factors.

Microchip-based capillary electrophoresis for determination of lactate dehydrogenase isoenzymes.

This article describes a novel microchip-based capillary electrophoresis and oncolumn enzymatic reaction analysis protocol for lactate dehydrogenase (LDH) isoenzymes with a home-made xenon lamp-induced fluorescence detection system. A microchip integrated with a temperature-control unit is designed and fabricated for low-temperature electrophoretic separation of LDH isoenzymes, optimal enzyme reaction temperature control, and product detection. A four-step operation and temperature control are employed for the determination of LDH activity by on-chip monitoring of the amount of incubation product of NADH during the fixed incubation period and at a fixed temperature. Experiments on the determination of LDH standard sample and serum LDH isoenzymes from a healthy adult donor are carried out. The results are comparable with those obtained by conventional CE. Shorter analysis times and a more stable and lower background baseline can be achieved. The efficient separation of different LDH forms indicates the potential of microfluidic devices for isoenzyme assay.

Numerical analysis of an electrokinetic double-focusing injection technique for microchip CE.

The injection techniques in electrophoresis microchips play an important role in the sample-handling process, whose characteristics determine the separation performance achieved, and the shape of a sample plug delivered into the separation channel has a great impact on the high-quality separation performance as well. This paper describes a numerical investigation of different electrokinetic injection techniques to deliver a sample plug within electrophoresis microchips. A novel double-focusing injection system is designed and fabricated, which involves four accessory arm channels in which symmetrical focusing potentials are loaded to form a unique parallel electric field distribution in the intersection of injection channel and separation channel. The parallel electric field effectuates virtual walls to confine the spreading of a sample plug at the intersection and prevents sample leakage into separation channel during the dispensing step. The key features of this technique over other injection techniques are the abilities to generate regular and nondistorted shape of sample plugs and deliver the variable-volume sample plugs by electrokinetic focusing. The detection peak in the proposed injection system is uniform regardless of the position of the detection probe in the separation channel, and the peak resolution is greatly enhanced. Finally, the double-focusing injection technique shows the flexibility in detection position and ensures improved signal sensitivity with good peak resolution due to the delivered high-quality sample plug.

[Establishment of a method with micro-channel electrophoresis for detecting the mutations of isoniazid-resistant genes in Mycobacterium tuberculosis].

OBJECTIVE: To establish a method for the detection of isoniazid resistance-associated mutations in katG gene and inhA gene in Mycobacterium tuberculosis with single-stranded conformation polymorphism (SSCP) analysis by the micro-channel electrophoresis chip system. METHODS: The polymer solutions of acrylamide and its derivatives were used for sieving matrices with small amount of acridine orange as the fluorescent tag. A novel pair of primers was designed to link an extra segment that could be base paired with the mutation region to the PCR products of wild type inhA gene, which made the single strands of wild type inhA fragments present a unique conformation. RESULTS: The wild type fragments of katG gene and the fragments with mutation at codon 315 can be distinguished, and the inhA fragments with wild type and mutated regulatory sequence can be distinguished by this method. Twenty-two out of the 23 resistant strains were detected from 30 clinical isolates, the efficiency being 95%. CONCLUSION: It is demonstrated the high speed and sensitivity in detecting the mutations of isoniazid-resistant genes in Mycobacterium tuberculosis by micro-channel electrophoresis, and this method may be applicable in clinical detection of isoniazid-resistant strains.

A signal process method for DNA segments separation in micro-channel electrophoresis.

This paper presents a signal process method for DNA segments separation in micro-channel electrophoresis. It is developed and optimized by using a laser induced fluorescence (LIF) based detection system. In this detection system, signal is sampled and processed through a novel signal process module. The results show that this signal process method provides good signal-to-noise ratios and lower limit of detection (LOD).