Fabricating New Miniaturized Biosensors for the Detection of Dna Damage and Dna Mismatches

A large number of genetic diseases and genetic disorders are simply caused by base alterations in the genome. Therefore, developing efficient and cost effective techniques for routine detection of these alterations is of great importance. Different methods involving gel electrophoresis and Polymerase Chain Reaction have been widely employed, but majority of these methods are costly, time consuming, and lack throughput, creating a fundamental gap between the current state-of-the-art and desired characteristics of low-cost, high-speed, simplicity, versatility, and potential for miniaturization. In this study, we attempt to bridge this gap by developing new sensing platforms to detect DNA base mismatches and DNA damage with higher throughput, better ease-of-use, and with the potential to be miniaturized for greater portability. Two electrochemical mismatch detection sensing platforms were developed. One uses the electrochemical reduction of trans-4-cinnamic acid diazonium tetrafluoroborate. The other takes advantage of the natural ability of MutS protein for single base mismatch recognition. Also, two DNA damage detection assays were developed and the first approach uses Atomic Force Microscopy to monitor minor DNA damage by labeling damaged sites with a biomarker. This site-specific biolabeling was achieved through well-established biotin-streptavidin chemistry. In the second approach, a new layer-by-layer biomolecular immobilization method was introduced and used to detect DNA chemical damage using electrochemical technique

Fabricating New Miniaturized Biosensors for the Detection of Dna Damage and Dna Mismatches

A large number of genetic diseases and genetic disorders are simply caused by base alterations in the genome. Therefore, developing efficient and cost effective techniques for routine detection of these alterations is of great importance. Different methods involving gel electrophoresis and Polymerase Chain Reaction have been widely employed, but majority of these methods are costly, time consuming, and lack throughput, creating a fundamental gap between the current state-of-the-art and desired characteristics of low-cost, high-speed, simplicity, versatility, and potential for miniaturization. In this study, we attempt to bridge this gap by developing new sensing platforms to detect DNA base mismatches and DNA damage with higher throughput, better ease-of-use, and with the potential to be miniaturized for greater portability. Two electrochemical mismatch detection sensing platforms were developed. One uses the electrochemical reduction of trans-4-cinnamic acid diazonium tetrafluoroborate. The other takes advantage of the natural ability of MutS protein for single base mismatch recognition. Also, two DNA damage detection assays were developed and the first approach uses Atomic Force Microscopy to monitor minor DNA damage by labeling damaged sites with a biomarker. This site-specific biolabeling was achieved through well-established biotin-streptavidin chemistry. In the second approach, a new layer-by-layer biomolecular immobilization method was introduced and used to detect DNA chemical damage using electrochemical technique

Eliminating Absorbing Interference Using The H-point Standard Addition Method: Case of Griess Assay in The Presence of Interferent Heme Enzymes Such As NOS

Standard calibration methods used to determine trace analytes usually yield significant deviations from the actual analyte value in the presence of interferents in the assay media. These deviations become of particular concern when the concentration of the analyte is low, and when the results are used to draw mechanistic or kinetic conclusions, for instance in enzyme structure-function studies. In these circumstances, the H-point standard addition method (HPSAM) provides superior precision and accuracy. This method is developed here for the case of the spectrophotometric Griess assay used to determine nitrite in various enzymology investigations, such as nitrite determination in studies of nitrite reductases (NiR), or when determining nitrite as a breakdown product of nitric oxide synthesized by NOS enzymes. The results obtained by HPSAM are contrasted with those of the traditional calibration method

Reductive Decomposition of A Diazonium Intermediate by Dithiothreitol Affects The Determination of NOS Turnover Rates

Accurate determination of nitrite either as such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure–function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectrophotometric assay based on the Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed

Probe Functionalization With A Rhop-3 Antibody: Toward A Rhop-3 Antigen Immunosensor For Detection of Malaria

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K3Fe(CN)6, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker

Cholesterol Levels and Activity of Membrane Bound Proteins: Characterization by Thermal and Electrochemical Methods

The long-term goal of this investigation is to study the effects of increased cholesterol levels on the molecular activity of membrane-bound enzymes such as nitric oxide synthase, that are critical in the functioning of the cardiovascular system. In this particular investigation, we used differential scanning calorimetry (DSC) and dielectric thermal analysis (DETA) to study the effect of added cholesterol on melting/recrystallization and dielectric behavior, respectively, of phosphatidylcholine (PC) bilayered thin films. We also used electrochemical methods to investigate the effect of added cholesterol on the redox behavior of the oxygenase domain of nitric oxide synthase as a probe embedded in the PC films. The results show that added cholesterol in the PC films seems to depress the molecular dynamics as indicated by lowered current responses in the presence of cholesterol as well as a slight increase of the transition temperature in the overall two-phase regime behavior observed in PC–cholesterol films. These results are rationalized in the context of the general DSC and DETA behaviors of the PC–chol films

Fabricating New Miniaturized Biosensors for the Detection of Dna Damage and Dna Mismatches

A large number of genetic diseases and genetic disorders are simply caused by base alterations in the genome. Therefore, developing efficient and cost effective techniques for routine detection of these alterations is of great importance. Different methods involving gel electrophoresis and Polymerase Chain Reaction have been widely employed, but majority of these methods are costly, time consuming, and lack throughput, creating a fundamental gap between the current state-of-the-art and desired characteristics of low-cost, high-speed, simplicity, versatility, and potential for miniaturization. In this study, we attempt to bridge this gap by developing new sensing platforms to detect DNA base mismatches and DNA damage with higher throughput, better ease-of-use, and with the potential to be miniaturized for greater portability. Two electrochemical mismatch detection sensing platforms were developed. One uses the electrochemical reduction of trans-4-cinnamic acid diazonium tetrafluoroborate. The other takes advantage of the natural ability of MutS protein for single base mismatch recognition. Also, two DNA damage detection assays were developed and the first approach uses Atomic Force Microscopy to monitor minor DNA damage by labeling damaged sites with a biomarker. This site-specific biolabeling was achieved through well-established biotin-streptavidin chemistry. In the second approach, a new layer-by-layer biomolecular immobilization method was introduced and used to detect DNA chemical damage using electrochemical technique

Eliminating Absorbing Interference Using The H-point Standard Addition Method: Case of Griess Assay in The Presence of Interferent Heme Enzymes Such As NOS

Standard calibration methods used to determine trace analytes usually yield significant deviations from the actual analyte value in the presence of interferents in the assay media. These deviations become of particular concern when the concentration of the analyte is low, and when the results are used to draw mechanistic or kinetic conclusions, for instance in enzyme structure-function studies. In these circumstances, the H-point standard addition method (HPSAM) provides superior precision and accuracy. This method is developed here for the case of the spectrophotometric Griess assay used to determine nitrite in various enzymology investigations, such as nitrite determination in studies of nitrite reductases (NiR), or when determining nitrite as a breakdown product of nitric oxide synthesized by NOS enzymes. The results obtained by HPSAM are contrasted with those of the traditional calibration method

Reductive Decomposition of A Diazonium Intermediate by Dithiothreitol Affects The Determination of NOS Turnover Rates

Accurate determination of nitrite either as such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure–function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectrophotometric assay based on the Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed

Probe Functionalization With A Rhop-3 Antibody: Toward A Rhop-3 Antigen Immunosensor For Detection of Malaria

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K3Fe(CN)6, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker