Determination of nucleic acids at nanogram level using resonance light scattering technique with Congo Red

Based on the enhancement of the resonance light scattering (RLS) of Congo Red (CR) by nucleic acid, a new quantitative method for nucleic acid is developed. In the Tris-HCl buffer (pH 10.5), the weak light scattering of CR is greatly enhanced by addition of nucleic acid and CTMAB, the maximum peak is at 560 nm and the enhanced intensity of RLS is in proportion to the concentration of nucleic acid. The linear range is 1.0 x 10(-9) to 1.0 x 10(-6) g ml(-1), 7.5 x 10(-8) to 1.0 x 10(-6) g ml(-1) and 7.5 x 10(-8) to 2.5 x 10(-6) g ml(-1) for herring sperm DNA, calf thymus DNA and yeast RNA, and the detection limits are 0.019, 0.89 and 1.2 ng ml(-1) (S/N = 3), respectively. Actual biological samples were satisfactorily determined.     

Interaction of morin-cetyltrimethylammonium bromide with nucleic acids and determination of nucleic acids at nanograms per milliliter levels based on the enhancement of preresonance light scattering

A new preresonance light scattering (PRLS) assay of nucleic acids is presented. At pH 7.30, the weak PRLS of morin-cetyltrimethylammonium bromide (CTMAB) can be greatly enhanced by the addition of nucleic acids, owing to the interaction between the nucleic acid and morin-CTMAB. After the addition of morin and CTMAB to DNA, the zeta potential of DNA decreases and changes from negative to positive, which is due to the formation of an associate, the aggregation of morin on nucleic acids and the electric neutralization between DNA and the cationic surfactant CTMAB. Mechanism studies showed that the enhanced PRLS comes from the aggregation of morin in the presence of nucleic acids and CTMAB. The enhanced intensity of PRLS is in proportion to the concentration of nucleic acids in the range 7.5 x 10(-9)-1.0 x 10(-5) g ml(-1) for calf thymus DNA, 7.5 x 10(-9)-1.0 x 10(-6) g ml(-1) for salmon sperm DNA and 1.0 x 10(-8)-1.0 x 10(-6) g ml(-1) for yeast RNA. The detection limits are 3.4, 6.2 and 4.1 ng ml(-1) for calf thymus DNA, salmon sperm DNA and yeast RNA, respectively. Synthetic samples were analyzed satisfactorily.     

Study on the resonance light scattering spectrum of berberine-cetyltrimethylammonium bromide system and the determination of nucleic acids at nanogram levels

The interaction of berberine with nucleic acid in the presence of cetyltrimethylammonium bromide (CTMAB) in aqueous solution has been studied by spectrophotometry and resonance light scattering (RLS) spectroscopy. At pH 7.30, the RLS signals of berberine were greatly enhanced by nucleic acid in the region of 300-600 nm characterized by four peaks at 324.0, 386.5, 416.5 and 465.0 nm. The binding properties were examined by using a Scatchard plot based on the measurement of enhanced RLS data at 416.5 nm. Under optimum conditions, the increase of RLS intensity of this system at 416.5 nm is proportional to the concentration of nucleic acid. The linear range is 7.5 x 10(-9)-7.5 x 10(-5) g ml(-1) for calf thymus DNA, 7.5 x 10(-9)-2.5 x 10(-5) g ml(-1) for herring sperm DNA, and 5.0 x 10(-9)-2.5 x 10(-5) g ml(-1) for yeast RNA. The detection limits (S/N = 3) are 2.1 ng ml(-1) for calf thymus DNA, 6.5 ng ml(-1) for herring sperm DNA and 3.5 ng ml(-1) for yeast RNA, respectively. Three synthetic samples were analyzed satisfactorily.     

The sensitive determination of nucleic acids using resonance light scattering quenching method

It is found that in hexamethylene tetramine (HMTA)-HCl buffer of pH 7.00, nucleic acids can quench the resonance light scattering (RLS) of europium (III) (Eu3+)-2-thenoyltrifluoroacetne (TTA)-1,10-phenanthroline (Phen) system. Based on this, a sensitive method for the determination of nucleic acids is proposed. The experiments indicate that under the optimum conditions, the quenched RLS intensity is in proportion to the concentration of nucleic acids in the range of 1.0x10(-10) to 2.0x10(-6) g ml-1 for fish sperm (fsDNA), 1.0x10(-11) to 1.0x10(-6) g ml-1 for yeast RNA (yRNA), 5.0x10(-11) to 5.0x10(-7) g ml-1 for calf thymus DNA (ctDNA). Their detection limits (S/N=3) are 0.03, 0.006 and 0.002 ng ml-1, respectively. Therefore, the proposed method is the most sensitive RLS method for the determination of nucleic acids so far. The interaction between nucleic acids and Eu3+-TTA-Phen is also discussed.     

Fluorescence enhancement of yttrium(III)-rutin by nucleic acids in the presence of cetyltrimethylammonium bromide

It is found that nucleic acids can enhance the fluorescence intensity of yttrium(III) (Y(3+))-rutin in presence of cetyltrimethylammonium bromide (CTMAB) system. In hexamethylenetetramine (HMTA)-HCl buffer, the maximum enhanced fluorescence is produced, with maximum excitation and emission wavelengths at 452 and 520 nm, respectively. Based on this, a new fluorimetric method of determination of nucleic acids is proposed. Under optimum conditions, the enhanced fluorescence intensity is proportion to the concentration of nucleic acids in the range of 1.0 x 10(-7) to 1.0 x 10(-5)g/ml for fish sperm DNA (fsDNA), 1.0 x 10(-7) to 4.6 x 10(-6)g/ml for yeast RNA (yRNA), their detection limits (S/N=3) are 7.5 x 10(-8), 8.0 x 10(-8)g/ml, respectively. The interaction mechanism is also studied.     

Fluorescence enhancement effect of morin-nucleic acid-L-cysteine-capped nano-ZnS system and the determination of nucleic acid

It is found that L-cysteine-capped nano-ZnS can further enhance the fluorescence intensity of the morin-nucleic acid system. Under optimum conditions, the enhanced intensity of fluorescence is proportional to the concentration of nucleic acid in the range of 7.0 x 10(-8)-1.0 x 10(-5) g mL(-1) for fish sperm DNA (fsDNA) and 9.0 x 10(-8)-5.0 x 10(-6) g mL(-1) for yeast RNA (yRNA). The corresponding detection limits (S/N = 3) are 2.0 x 10(-8) g mL(-1) and 4.0 x 10(-8) g mL(-1), respectively. The interaction mechanisms of morin-nucleic acid-L-cysteine-capped nano-ZnS system are studied by multiple techniques. It is considered that there exists synergistic effects of groove binding and electrostatic interaction between morin, L-cysteine-capped nano-ZnS and nucleic acid, and the complex of morin-L-cysteine-capped nano-ZnS-nucleic acid is formed.     

Study on the interaction between nucleic acids and cationic surfactants

The interactions of nucleic acids and cationic surfactants (cetylpyridine bromide (CPB) and cetyltrimethylammonium bromide (CTMAB)) in aqueous solution have been studied using the techniques of resonance light scattering (RLS) spectroscopy, the absorption spectroscopy, zeta potential assay and NMR assignment measurement. It is considered that CPB or CTMAB can assemble on the surface of nucleic acid via electrostatic and hydrophobic forces, which results in the formation of large associate of nucleic acid-cationic surfactant and RLS enhancement of nucleic acid. Besides these forces, the pi-pi stacking force between CPB and nucleic acid also exists in the associate. In comparison with CTMAB, CPB has larger enhancement on RLS of nucleic acid, which is attributed to that the enhancement of the former is only due to the absorption of the bases of nucleic acid, while the enhancement of the latter is own to the synergetic resonance caused by the absorption of both bases of nucleic acid and the pyridyl in CPB. These results have important implication for understanding the influence of surfactants on nucleic acid functionality in life science.     

Nucleic acids analysis with nano-Ag-Tb(III) by a resonance light scattering technique.

The nano-Ag-terbium(III)-mucleic acids system was observed by a resonance light scattering (RLS) technique for the first time, and the quantitative analysis of nucleic acids at nanogram levels was established. Studies showed that the RLS intensity of the nano-Ag-terbium(III) system can be obviously enhanced by nucleic acid, which was characterized by the RLS spectrum and the UV-Vis spectrum. In this system, the nanoparticles were only of a definite size and in a limited particle concentration region. Further research indicated that under the optimum conditions, the enhanced intensity of RLS is in proportion to the concentration of nucleic acids in the ranges of 7.0 x 10(-9) g ml(-1) to 8.0 x 10(-6) g ml(-1) for calf thymus DNA (ctDNA), 2.0 x 10(-8) g ml(-1) to 1.0 x 10(-6) g ml(-1) for fish sperm DNA (fsDNA) and 1.0 x 10(-9) g ml(-1) to 1.0 x 10(-7) g ml(-1) for yeast RNA (yRNA). The detection limits were 1.4 ng ml(-1) for ctDNA, 1.2 ng ml(-1) for fsDNA and 0.85 ng ml(-1) for yRNA, respectively. Synthetic and real samples were determined satisfactorily.     

Molecular spectroscopy study of the reaction of nucleic acids with brilliant cresol blue

The interaction of brilliant cresol blue (BCB) with nucleic acids in aqueous solution has been studied by spectrophotometry and Rayleigh light scattering (RLS) spectroscopy. Under suitable conditions, the RLS spectra of BCB changed significantly due to the presence of nucleic acids. RLS intensity of BCB at 364 nm is greatly enhanced with the addition of nucleic acids, and a new RLS peak is observed at 552 nm. This peak is about half the intensity of that at 364 nm. The results of this study show that BCB interacts with DNA possibly due to the cooperative effect of electrostatic attraction, intercalation, coordination and hydrophobic effect. Under optimum conditions, the increase of RLS at 364 nm of a BCB solution is proportional to the concentration of nucleic acids added. This result is the basis for a new RLS method for determination of nucleic acids. The linear range of ctDNA, fsDNA and yRNA is 0.12-4.70, 0.11-4.64 and 0.43-7.07 microg ml(-1), respectively.     

Application of aluminium(III) complex with salicylidene-o-aminophenol to the fluorometric determination of nucleic acids

In buffer medium of hexamethylene tetraamine-HCl at pH 5.9 the aluminium(III) complex with salicylidene-o-aminophenol (SAP) has a fluorescence peak at 508 nm with excitation at 410 nm. When nucleic acid coexists, it reacts with the complex within 8 min at room temperature to produce a non-fluorescent product, resulting in the decrease of fluorescence intensity of the aluminium complex. On basis of this, a new fluorometric method for nucleic acids determination is proposed. The calibration graphs for calf thymus DNA, fish sperm DNA and yeast RNA are linear up to 5.0, 4.0 and 3.0 microg ml(-1), respectively, and corresponding detection limits are 49, 52 and 62 ng ml(-1). The synthetic samples are analyzed with relative standard deviation of five measurements of 3.9-6.0%. DNA in an extraction product from human blood is determined using the calibration graph for calf thymus DNA, and the result is very close to that by the ethidium bromide assay. Compared with some established fluorometric methods, this procedure is sensitive, selective, reliable, reproducible and practical. The association constant of calf thymus DNA with the complex is estimated by two graphic methods. It is suggested that the binding reaction between nucleic acids with the complex proceeds in an intercalation way.     

Resonance Rayleigh scattering study of the reaction of nucleic acids with thionine and its analytical application

Resonance Rayleigh scattering (RRS) of the thionine (TH)-nucleic acids system and its analytical application have been studied. In pH 2.2 acidic buffer medium, some nucleic acids can react with TH to form TH-nucleic acids complex. This results in a great enhancement of RRS and the appearance of new RRS spectra. The RRS spectral characteristics of TH-ctDNA system, the affecting factors and the optimum conditions of the reaction have been investigated. The enhancement of the RRS signal is directly proportional to the concentration of nucleic acids in the range 0-10.0 microg/ml for calf thymus DNA and 0-15.0 microg/ml for yeast RNA, and its detection limits (3sigma) are 3.5 ng/ml for calf thymus DNA and 4.9 ng/ml for yeast RNA, respectively. The method shows a wide linear range and high sensitivity, and was applied to the determination of trace amounts of nucleic acid in synthetic samples and practical samples with satisfactory results. The bind properties for the interactions of TH with ctDNA were investigated using a Scatchard plot based on the measurement of the enhanced RRS data at 340 nm, and the binding number and intrinsic binding constant are 4.9 and 2.6 x 10(5) mol/dm(3), respectively.     

Determination for micro amounts of nucleic acids by a resonance light scattering technique with dequalinium chloride

Based on the strong enhancement effect of nucleic acids on resonance light scattering of dequalinium chloride, the determination method for micro amounts of nucleic acids has been developed. Under the experimental conditions (5.0x10(-5) mol l(-1) dequalinium, pH 7.0, at room temperature) the linear range of this assay is 0.04-10.0 mug ml(-1) for calf thymus DNA and fish sperm DNA, and 0.04-35.0 mug ml(-1) for yeast RNA. The detection limits (3sigma) are 6.2 ng ml(-1) for calf thymus DNA, 7.4 ng ml(-1) for fish sperm DNA, and 7.0 ng ml(-1) for yeast RNA, respectively. Almost no interference can be observed from ionic strength, proteins, nucleoside, and most of the metal ions. Six synthetic samples were determined satisfactorily.     

The interaction of poly(ethylenimine) with nucleic acids and its use in determination of nucleic acids based on light scattering

For the first time, poly(ethylenimine) (PEI) was used to determine nucleic acids with a light scattering technique using a common spectrofluorometer. The interaction of PEI with DNA results in greatly enhanced intensity of light scattering at 300 nm, which is caused by the formation of the big particles between DNA and PEI. Based on this, a new quantitative method for nucleic acid determination in aqueous solutions has been developed. Under the optimum conditions, the enhanced intensity of light scattering is proportional to the concentration of nucleic acid in the range of 0.01-10.0 microg ml(-1) for herring sperm DNA (hsDNA), 0.02-10.0 microg ml(-1) for calf thymus DNA (ctDNA), 0.02-20.0 microg ml(-1) for yeast RNA (yRNA). The detection limits are 5.3, 9.9, and 13.7 ng ml(-1), respectively. Synthetic samples were determined satisfactorily. At the same time, the light scattering technique has been successfully used to obtain the information on the effects of pH and ionic strength on the formation and the stability of the DNA/PEI complex, which is important in some fields such as genetic engineering and gene transfer. Using ethidium bromide (EB) as a fluorescent probe, the binding of PEI with hsDNA was studied. Both the binding constant of EB with DNA and the number of binding sites per nucleotide decrease with increasing concentration of PEI, indicating noncompetitive inhibition of EB binding to DNA in the presence of PEI. And the association constant of PEI to DNA obtained is 1.2 x 10(5) M(-1). IR-spectra show that PEI interacts with DNA through both the phosphate groups and the bases of DNA and the formation of DNA/PEI complex may cause the change of the conformation of the DNA secondary structure, which is also proved by UV-spectra.     

Determination of nucleic acids with crystal violet by a resonance light-scattering technique

For the first time, Crystal Violet (CV) was used to determine nucleic acid concentrations using the resonance light-scattering (RLS) technique. Based on the enhancement of the RLS of CV by nucleic acids, a new quantitative determination method for nucleic acids in aqueous solutions has been developed. At pH 5.03 and ionic strength 0.005 mol kg-1, the interaction of CV with nucleic acids results in three characteristic RLS peaks at 344.0, 483.0 and 666.0 nm. With 4.0 x 10(-5) mol l-1 of CV, linear relationships were found between the enhanced intensity of RLS at 666.0 nm and the concentration of nucleic acids in the range 0-2.5 micrograms ml-1 for herring sperm DNA, 0-4.0 micrograms ml-1 for calf thymus DNA and 0-4.5 micrograms ml-1 for yeast RNA. The limits of determination were 13.8 ng ml-1 for herring sperm DNA, 36.8 ng ml-1 for calf thymus DNA and 69.0 ng ml-1 for yeast RNA. The assay is convenient, rapid, inexpensive and simple.     

An ultrasensitive nucleic acid biosensor based on the catalytic oxidation of guanine by a novel redox threading intercalator

An ultrasensitive nucleic acid biosensor for the direct detection of attomoles nucleic acid in 1.0-5.0 [corrected] microl droplets is described which can be used for detection of cancer marker genes in mRNA extracted from human breast tissues without a RT-PCR step.     

Determination of chitosan by cathodic stripping voltammetry.

A sensitive method for the determination of chitosan (CTS) by cathodic stripping voltammetry is presented. The method exploits a pair of oxidation-reduction peaks of CTS at -0.62 V (vs. SCE) and -0.54 V (vs. SCE), and an enhancement of the peak current of CTS observed in a 0.05 mol l(-1) potassium hydrogenphthalate buffer solution (pH 2.5). The peak current is linear with the concentration of CTS from 5.0 x 10(-7) to 1.5 x 10(-5) g ml(-1), and the detection limit is 1.0 x 10(-7) g ml(-1). We studied the characteristics and the mechanism of the electrode reaction, which proved that this process was diffusion controlled. This method was applied to determine the content of CTS in real samples with satisfactory results.     

Study on the interaction between silver nanoparticles and nucleic acids in the presence of cetyltrimethylammonium bromide and its analytical application

A novel method is proposed in this paper, that is the silver nanoparticle (nanoAg)-cetyltrimethylammonium bromide (CTMAB) is used as the probe of resonance light scattering (RLS) for the determination of nucleic acids. Under optimum conditions, there are linear relationships between the quenching extent of RLS and the concentration of nucleic acids in the range of 4.0x10(-9)-2.0x10(-6)gmL(-1) for fish sperm DNA (fsDNA), 7.0x10(-9)-1.8x10(-6)gmL(-1) for calf thymus DNA (ctDNA) and 6.0x10(-9)-1.0x10(-6)gmL(-1) for yeast RNA (yRNA). The detection limits (S/N=3) of fsDNA, ctDNA and yRNA are 2.7x10(-10)gmL(-1), 4.8x10(-10)gmL(-1) and 7.2x10(-10)gmL(-1), respectively. The studies indicate that there are interactions among nanoAg, CTMAB and fsDNA through electrostatic and chemical affinity, and the nanoAg-CTMAB complex can induce the structural change of base stacking and helicity of fsDNA.     

Spectrofluorimetric method for determination of aluminium with chromotropic acid and its application to water samples

A rapid and sensitive method for the trace level determination of aluminium based on the formation of a 1:1 complex with chromotropic acid (1,8-dihydroxynaphthlene-3,6-disulfonic acid) in an methanol medium is reported. The fluorescence intensity of the system was 50 times greater than that of the system without aluminium. This method is very sensitive and selective for the direct determination of aluminium ion. The fluorescence is excited at 346 nm and measured at 370 nm. The optimum conditions are a chromotropic acid concentration of 5.0 ml (1.0x10(-4) M) and pH 4.0+/-0.5 (acetic acid-sodium acetate buffer). The fluorescence intensity is a linear function of the concentration of Al(III) in the range 2-100 ng ml(-1) and the detection limit is 1.0 ng ml(-1). The method has been applied successfully to the determination of trace amount of Al(III) in tap, river and sea-water samples.     

Automated flow-injection pseudotitration of strong and weak acids, ascorbic acid and calcium, and catalytic pseudotitrations of aminopolycarboxylic acids by use of a microcomputer-controlled analyser

A simple, inexpensive, fully automated spectrophotometric system for flow-injection pseudotitrations is used to perform acid-base, redox, complexometric and catalytic "titrations". Peak widths (in time units) in the range 10-100 sec can be measured with a precision of better than 0.3% in most cases. Strong and weak acids in the range 5.0 x 10(-4)-1.0 x 10(-2)M are measured by using sodium hydroxide-Bromothymol Blue "titrant". Ascorbic acid (1 x 10(-4)-1 x 10(-2)M) is "titrated" with 2,6-dichlorophenolindophenol, and calcium (5.0 x 10(-4)-5.0 x 10(-2)M) with EDTA, with calmagite as indicator in the presence of magnesium. Aminopolycarboxylic acids (5 x 10(-6)-1 x 10(-2)M) are measured by use of catalytic indication based on the manganese-catalysed periodate-diethylaniline reaction. The ascorbic acid method has been applied to analysis of pharmaceutical preparations, and the calcium method to water analysis.     

Resonance double light scattering method for the determination of proteins with morin-CTMAB

A new determination method of proteins with the limit of determination at nanogram levels is proposed by using a common spectrofluorimeter to detect intensity of resonance double line scattering (RDLS). Proteins including bovine serum albumin (BSA), human serum albumin (HSA) can combine with morin and cetyltrimethylammonium briomide (CTMAB) in the pH range 7.0-8.0 and produce enhanced RDLS signal at lambda(ex)/lambda(em) 305.0/610.0 nm. Optimization conditions for the morin-protein-CTMAB interaction were tested. In the studied system, BSA/CTMAB/morin = 1:2:3. The association constant of morin with BSA is 5.2 x 10(4). Under the optimum conditions, the linear range is 7.5 x 10(-8)-1.0 x 10(-5) g/ml for BSA, 2.5 x 10(-8)-5.0 x 10(-6) g/ml for HSA. The detection limits (S/N = 3) are 66.0 ng/ml for BSA and 23.0 ng/ml for HSA, respectively. Four synthetic samples were analyzed satisfactorily.