The Titrated Mannitol Improved Central [99mTc] Tc TRODAT-1 Uptake in an Animal Model-A Clinically Feasible Application.

[99mTc]Tc TRODAT-1 is a widely used single photon emission tomography (SPECT) radiopharmaceutical in Asian practice for early detection of central dopaminergic disorders. However, its imaging quality remains sub-optimal. To overcome this problem, mannitol, an osmotic agent was used to observe its effect on improving striatal [99mTc]Tc TRODAT-1 uptake in rat brain by titrated human dosages to investigate a clinically feasible way to improve human imaging quality. [99mTc]Tc TRODAT-1 synthesis and quality control were performed as described. Sprague-Dawley rats were used for this study. The animal in vivo nanoSPECT/CT and ex vivo autoradiography were employed to observe and verify the striatal [99mTc]Tc TRODAT-1 uptake in rat brains using clinically equivalent doses (i.e., 0, 1 and 2 mL groups, each n = 5) of mannitol (20% w/v, equivalent to 200 mg/mL) by an intravenous administration. Specific binding ratios (SBRs) were calculated to express the central striatal uptake in different experimental groups. In the NanoSPECT/CT imaging, the highest SBRs of striatal [99mTc]Tc TRODAT-1 were reached at 75-90 min post-injection. The averaged striatal SBRs were 0.85 ± 0.13 (2 mL normal saline, the control group), 0.94 ± 0.26 (1 mL mannitol group) and 1.36 ± 0.12 (2 mL mannitol group, p < 0.01 which were significantly different than the control as well as 1 mL mannitol groups (p < 0.05). The SBRs from ex vivo autoradiography also showed a comparable trend of the striatal [99mTc]Tc TRODAT-1 uptake in the 2 mL, 1 mL mannitol and the control groups (1.76 ± 0.52, 0.91 ± 0.29, and 0.21 ± 0.03, respectively, p < 0.05). No remarkable changes of vital signs were found in the mannitol groups and the controls. Pre-treated mannitol revealed a significant increase of the central striatal [99mTc]Tc TRODAT-1 uptake in a rat model which not only enabled us to perform pre-clinical studies of dopaminergic related disorders but also provided a potential way to further optimize image quality in clinical practice.

Rhesus Blood Typing within a Few Seconds by Packing-Enhanced Nanoscattering on Individual Erythrocytes.

A method for the ABO and Rhesus (Rh) blood group typing from individual erythrocytes is proposed in this study. Blood-group-specific antibodies immobilized to gold nanoparticles (BG-AuNP) were utilized for the identification of blood groups from individual erythrocytes by objective-type dark-field microscopy (OTDFM). The scattering of free BG-AuNP and their Brownian motion as well as BG-AuNP attached on erythrocytes is easily observed by OTDFM. The strong scattering intensity caused by BG-AuNP packing-enhanced nanoscattering (PENS) on erythrocytes is first demonstrated. PENS combined with OTDFM allows us to identify blood groups within 5 s for all blood group antigens including A, B, D, C, c, E, and e. This was immediately identified by mixing with BG-AuNP without any washing step or waiting for hemoagglutination. Therefore, PENS combined with OTDFM demonstrates feasibility and advantages for use in emergency transfusions where the blood group of patients is unknown. Moreover, matching RhD+ in the case of emergency transfusions may also be beneficial in reducing the shortage of RhD- red blood cell concentrate in the case of a population with a high frequency in RhD-.

Managing Vibration Training Safety by Using Knee Flexion Angle and Rating Perceived Exertion.

Whole-body vibration (WBV) is commonly applied in exercise and rehabilitation and its safety issues have been a major concern. Vibration measured using accelerometers can be used to further analyze the vibration transmissibility. Optimal bending angles and rating of perceived exertion (RPE) evaluations have not been sufficiently explored to mitigate the adverse effect. Therefore, the aims of this study were to investigate the effect of various knee flexion angles on the transmissibility to the head and knee, the RPE during WBV exposure, and the link between the transmissibility to the head and the RPE. Sixteen participants randomly performed static squats with knee flexion angles of 90, 110, 130, and 150 degrees on a WBV platform. Three accelerometers were fixed on the head, knee, and center of the vibration platform to provide data of platform-to-head and platform-to-knee transmissibilities. The results showed that the flexion angle of 110 degrees induced the lowest platform-to-head transmissibility and the lowest RPE (p < 0.01). A positive correlation between RPE and the platform-to-head transmissibility was observed. This study concluded that a knee flexion of about 110 degrees is most appropriate for reducing vibration transmissibility. The reported RPE could be used to reflect the vibration impact to the head.

Orthogonal Tip-to-Tip Nanocapillary Alignment Allows for Easy Detection of Fluorescent Emitters in Femtomolar Concentrations.

Here we present the realization of a novel fluorescence detection method based on the electromigration of fluorescent molecules within a nanocapillary combined with the laser excitation through a platinum (Pt)-coated nanocapillary. By using the Pt nanocapillary assisted focusing of a laser beam, we completely remove the background scattering on the tip of the electrophoretic nanocapillary. In this excitation geometry, we demonstrate a 1000-fold sensitivity enhancement (1.0 nM to 1.0 pM) compared to the detection in microcapillaries with epifluorescence illumination and fluorescence spectrophotometry. Due to a significant electroosmotic flow, we observe a decelerating migration of DNA molecules close to the tip of the electrophoretic nanocapillary. The reduced DNA translocation velocity causes a two-step stacking process of molecules in the tip of the nanocapillary and can be used as a way to locally concentrate molecules. The sensitivity of our method is further improved by a continuous electrokinetic injection of DNA molecules followed by sample zone stacking on the tip of the nanocapillary. Concentrations ranging from 0.1 pM to 1.0 fM can be directly observed on the orifice of the electrophoretic nanocapillary. This is a 1000-fold improvement compared to traditional capillary electrophoresis with laser-induced fluorescence.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target (HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

Recent Advances in the Determination of Pesticides in Environmental Samples by Capillary Electrophoresis.

Nowadays, owing to the increasing population and the attempts to satisfy its needs, pesticides are widely applied to control the quantity and quality of agricultural products. However, the presence of pesticide residues and their metabolites in environmental samples is hazardous to the health of humans and all other living organisms. Thus, monitoring these compounds is extremely important to ensure that only permitted levels of pesticide are consumed. To this end, fast, reliable, and environmentally friendly methods that can accurately analyze dilute, complex samples containing both parent substances and their metabolites are required. Focusing primarily on research published since 2010, this review summarizes the use of various sample pretreatment techniques to extract pesticides from various matrices, combined with on-line preconcentration strategies for sensitivity improvement, and subsequent capillary electrophoresis analysis.

Reversible Association of Nitro Compounds with p-Nitrothiophenol Modified on Ag Nanoparticles/Graphene Oxide Nanocomposites through Plasmon Mediated Photochemical Reaction.

Because localized surface plasmon resonance in nanostructures of noble metals is accompanied by interesting physical effects such as optical near-field enhancement, heat release, and the generation of hot electrons, it has been employed in a wide range of applications, including plasmon-assisted chemical reactions. Here, we use a composite of silver nanoparticles and graphene oxide (Ag@GO) as the catalytic as well as the analytic platform for plasmon-assisted chemical reactions. Through time-dependent surface-enhanced Raman scattering experiments, it is found that p-nitrothiophenol (pNTP) molecules on Ag@GO can be associated with nitro compounds such as nitrobenzene and 1-nitropropane to form azo compounds when aided by the plasmons. Furthermore, the reaction rate can be modulated by varying the wavelength and power of the excitation laser as well as the nitro compounds used. In addition, the aforementioned coupling reaction can be reversed. We demonstrate that the oxidation of azo compounds on Ag@GO using KMnO4 leads to the dissociation of the N═N double bond in the azo compounds and that the rate of bond dissociation can be accelerated significantly via laser irradiation. Furthermore, the pNTP molecules on Ag@GO can be recovered after the oxidation reaction. Finally, we demonstrate that the plasmon-assisted coupling reaction allows for the immobilization of nitro-group-containing fluorophores at specific locations on Ag@GO.

Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary electrophoresis with argon ion laser-induced fluorescence.

In this work, five fluorescent dyes (SYTO-9, SYBR Green I, SYBR Green II, SYBR Safe, and SYBR Gold) were used as both on-column and precolumn stains for total RNA analysis by CE-LIF with Ar ion laser excitation. In the on-column RNA stain, the SYTO-9 provided the highest fluorescence intensity and the lowest detectable concentration, as low as 10 pg/µL, while the SYBR Green II and SYBR Gold were adsorbed on the poly(ethylene oxide) thus affected the separation efficiency. As a precolumn stain, SYBR Gold was the most sensitive among the five dyes due to the strong affinity between the dye and RNA molecules. As a result, a single-cell quantity of RNA (10-30 pg per cell) could be detected by CE-LIF with precolumn staining by SYBR Gold. Because of the great savings of fluorescent dye using precolumn stain (one button dye may use for one million stain), this method is the best strategy for RNA staining in terms of cost-effectiveness and sensitivity.

Baseline separation of amino acid biomarkers of hepatocellular carcinoma by polyvinylpyrrolidone-filled capillary electrophoresis with light-emitting diode-induced fluorescence in the presence of mixed micelles.

Physiological amino acids (AAs) are important indices for monitoring various diseases, including cancer. This study proposes a polymer-based separation method in the presence of mixed micelles for the determination of AAs by capillary electrophoresis with light-emitting diode-induced fluorescence. The separation of 18 amino acid-cyano[f]benzoisoindoles (AA-CBIs) was successfully achieved using a solution of polyvinylpyrrolidone (PVP, 5% w/v, Mavg 1,300,000 Da). In addition, we demonstrated that mixed micelles composed of sodium dodecyl sulfate and isopropanol may affect the migration order of the AA-CBIs and greatly improve the speed of separation. With the exception of proline, 21 plasma AA-CBIs, including high isoelectric point AAs (lysine, ornithine, and arginine), were identified by using optimized separation conditions with minimal matrix effects. The results of this study demonstrated the distinct advantages of the proposed method, such as simplicity, high efficiency, and cost-effectiveness. This method has great potential for the diagnosis of several important diseases, including carcinomas, aminoacidopathies, and neurotransmission disorders.

Simultaneous separation of five major ribonucleic acids by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow: application to the rapid screening of 5S rRNA from ovarian cancer cells.

RNA integrity is important in RNA studies because poor RNA quality may impact downstream methodologies. This study proposes a rapid and cost-effective method for the determination of RNA integrity based on CE-LIF in the presence of electroosmotic flow. The proposed method uses poly(ethylene) oxide (Mavg=4,000,000 Da) as a sieving matrix for total RNA separation. Ethidium bromide (µg mL(-1)) was dissolved in a polymer solution as an interchelating dye for on-column fluorescent labeling. The 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA from the total human RNA extracted from the cells were fully separated using the proposed method. The lowest detectable concentration of total RNA achieved was 100 pg µL(-1) with a 6 min sample injection followed by on-column concentration. In addition, the temperature-induced degradation of total RNA was observed by CE-LIF. The electropherograms revealed more fragmentation of 28S and 18S rRNAs by temperature-induced hydrolysis compared with the 5.8S rRNA, 5S rRNA and tRNA. Therefore, the results indicated that RNA degradation should be considered for long-term, high-temperature incubations in RNA-related experiments involving RNA hybridization. The proposed method is furthermore, applied to the determination of 5S rRNA overexpressed in ovarian cancer cells as compared to the cervical cancer cells. Overall, CE-LIF is highly promising for rapid screening of ovarian cancers without tedious pre-amplification steps.

Rapid screening of the heterogeneity of DNA methylation by single-strand conformation polymorphism and CE-LIF in the presence of electro-osmotic flow.

DNA methylation is a complex event in epigenetic studies because of both the large CpG islands present upstream of the promoter region and the different distribution of DNA methylation despite similar methylation levels. For this reason, we proposed a fast, cost-effective method for the screening of DNA methylation based on SSCP and CE-LIF. In this study, the PCR products that were amplified from bisulfite-treated genomic DNA were denatured at 94°C, followed by immediate chilling in ice water to form the ssDNA. The ssDNA were separated by 1.5% poly(ethylene oxide) (Mavg 8 000 000 Da) in the presence of EOF according to the different conformations represented by their unique methylation states. This result demonstrated that four hepatocellular carcinoma cell lines represented a different heterogeneity of DNA methylation and could be distinguished by SSCP-CE. The results obtained from SSCP-CE also corresponded with those obtained from combined bisulfide restriction analysis and methylation-sensitive high-resolution melting analysis. Therefore, the proposed SSCP-CE method may potentially be used for rapid screening for determination of the heterogeneity of DNA methylation in further epigenetic studies and clinical diagnosis.

Colorimetric determination of DNA methylation based on the strength of the hydrophobic interactions between DNA and gold nanoparticles.

A simple, novel colorimetric nanosensor for DNA methylation based on the strength of hydrophobic interaction between DNA and gold nanoparticles was proposed. The nanosensing of oligonucleotides with four nitrogen bases was first demonstrated by dividing the bases into two groups (A/T and C/G) using the representative colors that correspond to Watson-Crick base pairing. By treatment of the genomic DNA with sodium bisulfite followed by PCR amplification, the methylation level of nasopharyngeal carcinoma cells treated with 5-aza-2'-deoxycytidine for up to 5 days could be discriminated by naked eye observation. Furthermore, 12 cancer cell lines that demonstrate heterogeneity with respect to DNA methylation could also be distinguished using the nanosensor, even for amplicons as long as 342 bp. These results demonstrate that the proposed colorimetric nanosensor could potentially be useful in epigenetic studies.

Separation of amino acids by capillary electrophoresis with light-emitting diode-induced fluorescence in the presence of electroosmotic flow.

In this chapter, we describe a method to identify amino acids (AA) by capillary electrophoresis in conjunction with light-emitting diode-induced fluorescence (LEDIF). First, amino acids labeled with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide are converted to highly fluorescent cyanobenz[f]isoindole (CBI) derivatives. Next, they are separated by gel electrophoresis in the presence of EOF. In the process, the CBI products were excited by a violet LED to produce green fluorescence. In addition to the optical setup of light-emitting diode-induced fluorescence, the preparation of poly(ethylene) oxide for amino acid separation is also described in this chapter.

Comparative microRNA detection from precursor-microRNA-transfected hepatocellular carcinoma cells by capillary electrophoresis with dual-color laser-induced fluorescence.

A dual-LIF (dLIF) setup combined with CE for microRNA (miRNA) detection is proposed in this study. An argon ion laser (488 nm) and a solid state laser (640 nm) were chosen to excite the fluorescent dye-labeled DNA probe after splinted ligation of miRNA. The crosstalk of emission spectrum of Alex Fluor 488 and Alex Fluor 647 is minimized with a zero crosstalk matrix for Alex Fluor 647 to 488 channels. The linear ranges of the device for the fluorescent dye-labeled DNA probe were both from 1.0 nM to 0.1 pM. The limits of detection for Alexa Fluor 488-labeled DNA and Alex Fluor 647-labeled DNA were 9.3 and 31 fM, respectively. The detection of specific miRNA has been accomplished by combining splinted ligation with the fluorescent dye-labeled oligonucleotides. The linear range for the synthetic miRNA is from 1.0 nM to 1.0 pM. Without PCR amplification, CE-dLIF was applied to discriminate a pre-miR-10b*-transfected cells (contains precursor miR-10b*) from hepatocellular carcinoma cell (control cells). Therefore, this result indicates CE-dLIF has great potential to provide a rapid comparative assay for miRNAs detection.

Determination of RNA degradation by capillary electrophoresis with cyan light-emitted diode-induced fluorescence.

RNA integrity plays an important role in RNA studies because poor RNA quality may have a great impact on downstream methodologies. This study proposes a cost-effective, rapid, and sensitive method for determining RNA integrity based on capillary electrophoresis that utilizes a cyan light-emitted diode-induced fluorescence as a separation tool. The capillary was initially coated with 0.1% Poly(vinylpyrrolidone) (M(ave) 1,300,000 Da) to reduce electroosmotic flow and avoid RNA adsorption. When the capillary was filled with 0.4% poly(ethylene) oxide (M(ave) 4,000,000) and a nucleic acid-specific fluorescent dye, SYTO 9, the baseline separation of the 18S and 28S ribosomal RNAs (rRNAs) in total RNA was accomplished within 15 min. The lowest detectable concentration for the 18S and 28S rRNAs was estimated to be 50 pg/µL. Some peaks longer than the 28S rRNA that migrated slowly were observed as long as the initial total RNA concentration was optimized. The temperature-induced degradation of the large RNA fragments (longer than the 28S rRNA) was faster than that of 18S rRNA and 28S rRNA. These large RNA fragments may serve as a promising marker for testing RNA integrity compared to the traditional method.

Determination of the heterogeneity of DNA methylation by combined bisulfite restriction analysis and capillary electrophoresis with laser-induced fluorescence.

The methylation of the promoter region of DNA is an important regulatory mechanism for the downstream gene expression, and the extent of methylation has been linked to cancer formation. In this study, we report a simple method to screen for the degree of DNA methylation by combined bisulfite restriction analysis (COBRA) and capillary electrophoresis with laser-induced fluorescence (CE-LIF). After treating genomic DNA with sodium bisulfite, nested-PCR amplification and endonuclease (Taq I) digestion were performed. The digested DNA fragments were then separated by capillary electrophoresis using 1.5% poly(ethylene) oxide (M(ave), 8,000,000 g/mol) in the presence of electroosmotic flow. The improvement for DNA amplification using the nested PCR described here corresponded to approximately ten cells. In addition, the level of DNA methylation shown in the electropherograms obtained corresponded to the original percentage of DNA methylation from commercial available standard sample (0-100%). The electrophoretic patterns demonstrated that the six cancer cell lines tested displayed different degrees of DNA methylation and could be differentiated by hierarchical cluster analysis. Furthermore, the DNA methylation level was eliminated after treating the cells with an anti-cancer drug (5'-aza-2'-deoxycytidine). Together, these results suggest that CE-LIF is a potentially useful and cost-effective tool for cancer diagnosis or prognosis based on the heterogeneity in a patient's DNA.

Multiplexed microRNA detection by capillary electrophoresis with laser-induced fluorescence.

In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay's linear range covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N=3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs.

Quantitation of branched-chain amino acids in ascites by capillary electrophoresis with light-emitting diode-induced fluorescence detection.

Branched-chain amino acids (BCAAs) are one of the important biomarkers for monitoring liver disease such as hepatitis or hepatoma. In this communication, we present the determination of the concentrations of BCAA in ascites by CE light-emitted diode-induced fluorescence (LEDIF) using 1.5% m/v poly(ethylene oxide) (average M(v) : ~8 000 000 g/mol) that was prepared in 10 mM sodium tetraborate solution (pH 9.3). Naphthalene-2,3-dicarboxaldehyde was used to derivatize 15 amino acids (AAs) to form naphthalene-2,3-dicarboxaldehyde (NDA)-AA derivatives prior to CE analysis. The separation of 15 NDA-AA derivatives was accomplished within 15 min, with RSD values of <5.8% (within-day) and 7.4% (between-days) with respect to their migration times. The limits of detection for the tested BCAAs ranged from 10.6 to 10.9 nM. We determined the concentrations of three BCAAs--leucine, isoleucine and valine--in ascites by applying a standard addition method, with recovery percentages ranging from 93.9 to 111%. The results obtained from this CE-LEDIF method is in good agreement with those by a gold standard method using an AA analyzer. We have found that the concentrations of the three BCAAs in ascites obtained from patients suffering from liver diseases were lower than those from healthy individuals. Our approach is highly efficient, sensitive, and cost-effective, which holds great potential for the diagnosis of liver diseases.

Analysis of BART7 microRNA from Epstein-Barr virus-infected nasopharyngeal carcinoma cells by capillary electrophoresis.

MicroRNAs (miRNAs) are a class of approximately 22-nucleotide noncoding RNA molecules that negatively regulate their target genes in a sequence-specific manner. In the present study, a fluorescence-labeled antisense DNA oligonucleotide was directly hybridized with BART7 miRNA in SSC buffered-cetyltrimethylammonium bromide (CTAB), followed by capillary electrophoresis with laser-induced fluorescence. The CTAB-mediated hybridization allows the probe to anneal the target at 50.0 degrees C, which is well below the computer-calculated melting temperature of 66.4 degrees C. The free probe (22-nt) and probe/miRNA duplex (22-bp) can be separated well by 2% poly(ethylene) oxide in the presence of electroosmotic flow with 7 M urea. The repeatability of the migration time of the DNA probe was 10.66 +/- 0.34 min (n = 10), the resolution was 1.12 +/- 0.11 (n = 10), and the separation efficiencies achieved were 1.71 and 1.74 million per meter. The peak area of the probe/miRNA duplex exhibited an excellent linearity (r(2) = 0.9973). Furthermore, no false positive result was detected even in the presence of a 2000-fold excess of single nucleotide-mismatched target. Compared to other methods, capillary electrophoresis not only exhibits excellent specificity but also shows negligible effects of intrinsic interferences such as human total RNA, primary miRNA or precursor miRNA.

Identification of individual DNA molecule of Mycobacterium tuberculosis by nested PCR-RFLP and capillary electrophoresis.

The improvement of sensitivity and differentiation in rapidly identifying a small amount of mycobacteria in sputum has significant implications for reducing tuberculosis transmission. We previously applied the conventional PCR and capillary electrophoresis (CE) to establish the restriction fragment length polymorphism (RFLP) pattern of mycobacterial 65-kDa heat shock protein (hsp65) gene from colony specimens. However, the previous analysis did not provide enough sensitivity for sputum specimens in which the limitation of analysis might be hindered by PCR inhibitors and primer-dimers formation during amplification. In the current study, nested PCR (nPCR) had been redesigned for PCR-RFLP analysis (PRA) of mycobacterial hsp65 gene using CE. The results show both Mycobacterium tuberculosis complex and mycobacteria other than tuberculosis could be identified in the presence of PCR inhibitors. The interference due to primer-dimers was also minimized. Based on the Poisson distribution, the repeatability of single DNA molecule detection was greatly affected by sampling probability and might be improved significantly by increasing the sample loading. The PRA using nPCR and CE is not only able to detect the individual mycobacterial DNA molecule but also potentially differentiate the species.