Signal amplification based on DNA hybridization-dehybridization reaction on the surface of magnet submicrobeads for ultrasensitive DNA detection.

A new signal amplification strategy based on DNA hybridization-dehybridization reaction on the surface of magnetic submicrobeads (MSBs) for fluorescence detection of ultrasensitive DNA was developed. In this strategy, MSBs modified with probe DNA (DNAp-MSBs) were bound to target DNA (t-DNA) (with a ratio of 1 : 1) captured to a substrate. The DNAp-MSBs were released from the substrate via DNA dehybridization and then hybridized with Cy5-labeled detection DNA (Cy5-DNAd). After the Cy5-DNAd and DNAp-MSBs were separated by dehybridization, the Cy5-DNAd was collected. The DNAp-MSBs were then hybridized with other Cy5-DNAd to initiate the next hybridization-dehybridization round. This recycling of the hybridization-dehybridization process on the surface of the DNAp-MSBs was repeated multiple times to accumulate Cy5-DNAd. Finally, fluorescence intensity of the collected Cy5-DNAd was measured. Using this strategy, the limit of detection for determination of t-DNA was 8.5 × 10(-15) mol L(-1) for 11 cycles. The ultrasensitive assay was used to quantify ribosomal protein, large, P2 (RPLP2) mRNA in human breast cancer cells.

An ultra-sensitive DNA assay based on single-molecule detection coupled with hybridization accumulation and its application.

An ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation was developed. In this assay, target DNA (tDNA) in solution was accumulated on a silanized substrate blocked with ethanolamine and bovine serum albumin (BSA) through a hybridization reaction between tDNA and capture DNA immobilized on the substrate. The tDNA on the substrate was labeled with quantum dots which had been modified with detection DNA and blocked with BSA. The fluorescence image of single QD-labeled tDNA molecules on the substrate was acquired using total internal reflection fluorescence microscopy. The tDNA was quantified by counting the bright dots on the image from the QDs. The limit of detection of the DNA assay was as low as 6.4 × 10(-18) mol L(-1). Due to the ultra-high sensitivity, the DNA assay was applied to measure the beta-2-microglobulin messenger RNA level in single human breast cancer cells without a need for PCR amplification.

Ultrasensitive electrochemical DNA assay based on counting of single magnetic nanobeads by a combination of DNA amplification and enzyme amplification.

An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1-DNA-MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA-MNBs are released from the substrate via dehybridization. The released p1-DNA-MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin-AP conjugates. The resultant AP-p2-DNA-p1-DNA-MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 degrees C. AP on the AP-p2-DNA-p1-DNA-MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP-p2-DNA-p1-DNA-MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 x 10(-16) to 1.0 x 10(-13) mol/L.

Quantitative counting of single fluorescent molecules by combined electrochemical adsorption accumulation and total internal reflection fluorescence microscopy.

We developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat anti-rat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6- CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope coverslip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40 x 40 microm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying charge-coupled device. One hundred images of the fluorescent molecules accumulated on the coverslip were taken consecutively, one by one, by moving the coverslip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5 x 10(-15) to 5 x 10(-12) mol/L for R6G, 3 x 10(-15) to 2 x 10(-12) mol/L for IgG(H+L)-488, and 3 x 10(-15) to 2 x 10(-12) mol/L for DNA-R6G.

Reversion of P-glycoprotein-mediated multidrug resistance in human leukemic cell line by carnosic acid.

One of the common hindrances to successful chemotherapy is the development of multidrug resistance (MDR) by tumor cells to multiple chemotherapeutic agents. In this regard, P-glycoprotein (P-gp) acts as an energized drug pump that reduces the intracellular concentration of drugs, even of structurally unrelated ones. The modulators of P-gp function can restore the sensitivity of MDR cells to anticancer drugs. Therefore, to develop effective drug-resistance-reversing agents, we evaluated the P-gp modulating potential of carnosic acid (CA) in multidrug-resistant K562/AO2 cells in the present study. The reversing effect of CA was evaluated by determining the inhibition rates of cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assays. The intracellular adriamycin fluorescence intensity and the expression of P-gp were measured by flow cytometry (FCM). Meanwhile, the subcellular distribution of adriamycin was detected via Laser Scanning Confocal Microscopy (LSCM). The mRNA expression of mdrlwas then detected via semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The findings showed that CA decreased apparently the Inhibition Concentration 50% (IC50) of adriamycin by increasing its intracellular concentration and thus enhancing the sensitivity of K562/AO2 cells. Adriamycin was distributed evenly in the cytoplasm when the cells were treated with CA. The expression of mdrl was decreased. Overall, the results indicated that CA can serve as a novel, non-toxic modulator of MDR, and it can reverse the MDR of K562/AO2 cells in vitro by increasing intracellular adriamycin concentration, down-regulating the expression of mdrl, and inhibiting the function of P-gp.

Single-cell analysis by intracellular immuno-reaction and capillary electrophoresis with laser-induced fluorescence detection.

A novel method for single-cell analysis was developed by combining electroporation for intracellular immuno-reaction and capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Human interferon-gamma (IFN-gamma) in natural killer (NK) cells was chosen as the test antigen. Two forms of IFN-gamma in single cells could be well separated and detected with a limit of detection of zeptomole. In this assay, the anti-IFN-gamma monoclonal antibody labeled with fluorescein isothiocyanate (Ab*) was introduced into NK cells by electrophoration for intracellular immuno-reaction. After completion of the intracellular immuno-reaction, the NK cells were chemically pre-perforated with digitonin to lyse easily. Then, one NK cell containing the complexes of IFN-gamma isoantigens with Ab* was electrokinetically injected into the capillary. The cell adsorbed on the tip of capillary was lysed by ultrasonication. Finally, the complexes of the different forms of IFN-gamma in the cell were separated and detected by CE-LIF detection.

Determination of lactate dehydrogenase isoenzymes in single rat glioma cells by capillary electrophoresis with electrochemical detection.

A method for determination of lactate dehydrogenase (LDH) isoenzymes in single rat glioma cells (C6) was developed. In this method, a whole cell was electrokinetically injected into the front end of the separation capillary. After that, the cell was lysed by ultrasonication and the isoenzymes in the cell were pre-separated at 20 kV for 5 min and then incubated for 2 min with the enzyme substrates nicotinamide adenine dinucleotide (NAD(+)) and lactate in the capillary electrophoresis running buffer. The electroactive product NADH generated by the isoenzymes through on-capillary enzyme-catalyzed reaction was detected at the outlet of capillary by using the end-capillary amperometric detection with a constant potential mode at a carbon fiber bundle microdisk electrode. Since the amplification of signal via the enzyme reaction, the concentration of nicotinamide adenine dinucleotide (NADH) is much higher than that of LDH. The external standardization was used to quantify isoenzymes in individual cells. Three LDH isoenzymes in single rat glioma cells (C6) were determined and quantified.

Single-cell analysis by electrochemical detection with a microfluidic device.

A novel electrochemical method with a microfluidic device was developed for analysis of single cells. In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intracellular species were integrated in a microfluidic chip with a double-T injector coupled with an end-channel amperometric detector. A single cell was loaded at the double-T injector on the microfluidic chip by using electric field. Then, the docked cell was lysed by a direct current electric field strength of 220 V/cm. The analyte of interest inside the cell was electrokinetically transported to the detection end of separation channel and was electrochemically detected. External standardization was used to quantify the analyte of interest in individual cells. Ascorbic acid (AA) in single wheat callus cells was chosen as the model compound. AA could be directly detected at a carbon fiber disk bundle electrode. The selectivity of electrochemical detection made the electropherogram simple. The technique described here could, in principle, be applied to a variety of electroactive species within single cells.

Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination.

We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 µm and each spot has a diameter of ∼300 µm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3×10(-16) mol L(-1). The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of cDNAs corresponding to beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein, large, P2 mRNAs in single human breast cancer cells and 5 random synthetic DNAts are simultaneously quantified to examine the SMA and SMA-based single-cell multiple gene expression analysis.

Multiplexed optical coding nanobeads and their application in single-molecule counting analysis for multiple gene expression analysis.

A method for fabrication of multiplexed optical coding nanobeads (MOCNBs) was developed by hybridizing three types of coding DNAs labeled with different dyes (Cy5, FAM and AMCA) at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated nanobeads with a diameter of 300 nm. The color of the MOCNBs could be observed by overlapping three single-primary-color fluorescence images of the MOCNBs corresponding to emission of Cy5 (red), FAM (green) and AMCA (blue). The MOCNBs could be easily identified under a conventional fluorescence microscope. The MOCNBs with different colors could serve as the multiplexed optical coding labels for single-molecule counting analysis (SMCA) and be used in multi-gene expression analysis (MGEA). In the SMCA-based MGEA technique, multiple messenger RNAs (mRNAs) in cells could be simultaneously quantified through their complementary DNAs (cDNAs) by counting the bright dots with the same color corresponding to the single cDNA molecules labeled with the MOCNBs. We measured expression profiles of three genes from Lepidoptera insect Helicoverpa armigera in ∼100 HaEpi cells with and without steroid hormone inductions to demonstrate the SMCA-based MGEA technique using MOCNBs.

Carbon fiber bundle-Au-Hg dual-electrode detection for capillary electrophoresis.

The assembly of a dual-electrode amperometric detection system for capillary electrophoresis is developed. The dual-electrode detector consists of different kinds of electrode material (carbon fiber and Au-Hg). The dual-electrode is placed on the outlet end of the separation capillary. Cysteine, glutathione, ascorbic acid and uric acid can be detected simultaneously and selectively at the two electrodes of the dual electrode, respectively. The capillary electrophoresis-dual-electrode detection system has be used to determine these compounds in human blood samples.

Capillary electrophoretic enzyme immunoassay with electrochemical detection for cortisol.

A novel capillary electrophoretic enzyme immunoassay with electrochemical detection has been developed and used for the determination of cortisol. In this method, after the competitive enzyme immunoreaction, the free enzyme (horseradish peroxidase, HRP)-labeled cortisol (HRP-cortisol) and the bound enzyme-labeled cortisol (HPR-cortisol-anti-cortisol) were separated in the separation capillary and then catalyzed the enzyme substrate [3,3,5,5-tetramethyl-benzidine dihydrochloride, TMB(Red)] in the reaction capillary. The product of the enzymatic catalysis reaction [TMB(Ox)] was amperometrically detected on a carbon fiber microdisk bundle electrode. A concentration limit of detection (LOD) of 1.7 x 10(-1) mol/l, which corresponds to a mass LOD of 7.8 amol, was achieved with the relative standard deviation of 3.3%. The method has been verified using the cortisol controls.

Method of intracellular naphthalene-2,3-dicarboxaldehyde derivatization for analysis of amino acids in a single erythrocyte by capillary zone electrophoresis with electrochemical detection.

A novel method of intracellular derivatization was developed. In this method, the derivatization reagents [naphthalene-2,3-dicarboxaldehyde (NDA) and CN-] were introduced into living cells by electroporation for the derivatization reaction. After completion of derivatization reaction in cells, a single cell was drawn into the capillary tip by electroosmotic flow. Then the lysing solution was introduced into the capillary by diffusion. Once the individual cell was lysed, the derivatized amino acids in the individual cell were separated by capillary zone electrophoresis and detected by end-column amperometric detection at the outlet of the capillary. This method of intracellular NDA derivatization confined the analytes and the derivatization reagents to the volume of a single cell expanded. For an 8-microm erythrocyte, the contents were diluted by a factor of only ca. 1.6. The method was used to determination of amino acids in single erythrocytes. Six amino acids were identified and quantified.

Amperometric detection of perphenazine at a carbon fiber micro-disk bundle electrode by capillary zone electrophoresis.

A simple method for determination of perphenazine by capillary zone electrophoresis with amperometric detection is described. The optimum conditions of separation and detection are 1.50 x 10(-3) mol/l Na(2)B(4)O(7)-1.0 x 10(-3) mol/l NaOH (pH 9.9) for the buffer solution, 18 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, and 0.80 V versus saturated calomel electrode for the detection potential, respectively. The limit of detection is 5.0 x 10(-8) mol/l or 44 amol (S/N=3). The linear range of the calibration curve is 1.00 x 10(-7) to 1.00 x 10(-4) mol/l. The relative standard deviation is 1.5% for the migration time and 2.9% for the electrophoretic current at peak maximum. The method is applied to the determination of perphenazine in human urine.

Determination of midecamycin by capillary zone electrophoresis with electrochemical detection.

Capillary zone electrophoresis was employed for the determination of midecamycin using an end-column amperometric detection with a carbon fiber micro-disk bundle electrode at a constant potential of +1.15 V vs. saturated calomel electrode. The optimum conditions of separation and detection are 1.00x10(-3) mol l(-1) Na(2)HPO(4)-3.49x10(-4) mol l(-1) NaOH (pH 11.4) for the buffer solution, 20 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, respectively. The limit of detection is 5.0x10(-7) mol l(-1) or 0.41 fmol (S/N=3). The linear range of the calibration curve is 1.00x10(-6)-1.00x10(-3) mol l(-1). The relative standard deviation is 1.4% for the migration time and 4.9% for the electrophoretic peak current. The method could be applied to the determination of midecamycin in human urine. In this case, a separation voltage of 14 kV was used.

Capillary electrophoretic enzyme immunoassay with electrochemical detection using a noncompetitive format.

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) using a noncompetitive format has been developed. In this method, antigen (Ag) reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ag-Ab* complex produced in the solution are separated by capillary zone electrophoresis in a separation capillary. Then they catalyze enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB(Red)) and H(2)O(2) in a reaction capillary following the separation capillary. The reaction product, TMB(Ox), can be determined using amperometric detection on a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, a significant amount of TMB(Ox) can be produced for detection. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. A tumor marker (CA15-3) was used as a model, in order to test the method. The concentration LOD of CA15-3 is 0.024 U/ml, which corresponds to a mass detection limit of 1.3x10(-7) U.

Measurement of histamine in individual rat peritoneal mast cells by capillary zone electrophoresis with electrochemical detection.

Capillary zone electrophoresis was employed for the analysis of histamine in single rat peritoneal mast cells using an amperometric detector with a carbon fiber microdisk bundle electrode. In this method, individual mast cells and then 0.02 mol/l NaOH as a lysing solution are injected into the front end of the separation capillary by electromigration with an aid of a inverted microscope. A cell injector was constructed. Using it, the cell suspension was static, when a voltage for injecting single cells was applied. Histamine in single rat peritoneal mast cells have been identified. Quantitation has been accomplished through the use of calibration curves. The mean amount of histamine for nine cells is 95.8 fmol, which is consistent with the literature value.

Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection.

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.

Direct amperometric determination of lactate at a carbon fiber bundle microdisk electrode by capillary zone electrophoresis.

Capillary zone electrophoresis was employed for the determination of lactate using end-column amperometric detection at a carbon fiber bundle microdisk electrode. The optimum conditions of separation and detection are 3.6 x 10(-3) mol/l Na(2)HPO(4)-1.4 x 10(-3) mol/l NaH(2)PO (pH 7.2) for the buffer solution, 18 kV for the separation voltage and 1.60 V versus the saturated calomel electrode for the detection potential. The limit of detection is 7.6 x 10(-7) mol/l or 1.7 fmol (S/N=3) and the linear range is 1.7 x 10(-6)-8.2 x 10(-4) mol/l for the injection voltage of 6 kV and injection time of 5 s. The RSD is 1.8% for the migration time and 3.3% for the electrophoretic peak current. The method was applied to the determination of lactate in human saliva. The recovery of the method is between 95 and 109%.

Determination of glutathione in single human hepatocarcinoma cells by capillary electrophoresis with electrochemical detection.

A method for determination of glutathione (GSH) in single human hepatocarcinoma (HH) cells was described by capillary zone electrophoresis with electrochemical detection at a gold/mercury amalgam micro-disk electrode. When HH cells were washed with the running buffer instead of physiological buffer saline, only one electrophoretic peak for GSH is depicted on the electropherograms of single HH cells. When electroosmotic injection of 0.01 mol/l NaOH for lysing the cell introduced into the capillary, the lysis time can be shorten to 5 s. The whole cell injection and no need of derivatization reaction lead more accurate and precise results. The average amount of GSH in an individual HH cell is 22.3+/-5.8 fmol (mean+/-standard deviation), which is consistent with that of its homogenate.