Binding-Induced DNA Assembly and Its Application to Yoctomole Detection of Proteins

We describe the binding-induced DNA assembly principle and strategy that enable ultrasensitive detection of molecular targets and potential construction of unique nanostructures/nanoreactors. Two DNA motifs that are conjugated to specific affinity ligands assemble preferentially only when a specific target triggers a binding event. The binding-induced assembly of the DNA motifs results in the formation of a highly stable closed-loop structure, raising the melting temperature (<i>T</i>_m) of the hybrid by >30 °C and enabling effective differentiation of the target-specific assembly from the background. The ability to detect as few as a hundred molecules (yoctomole) of streptavidin, platelet derived growth factor, and prostate specific antigen represents an improvement of detection limits by 10³–10⁵-fold over traditional immunoassays. The assay is performed in a single tube, eliminating separation, immobilization, and washing steps of conventional assays. By incorporating unique signaling and structural features into the DNA motifs, we envision diverse applications in biosensing and nanotechnology

Aptamer-Modified Monolithic Capillary Chromatography for Protein Separation and Detection

A capillary chromatography technique was developed for the separation and detection of proteins, taking advantage of the specific affinity of aptamers and the porous property of the monolith. A biotinylated DNA aptamer targeting cytochrome c was successfully immobilized on a streptavidin-modified polymer monolithic capillary column. The aptamer, having a G-quartet structure, could bind to both cytochrome c and thrombin, enabling the separation of these proteins from each other and from the unretained proteins. Elution of strongly bound proteins was achieved by increasing the ionic strength of the mobile phase. The following proteins were tested using the aptamer affinity monolithic columns: human immunoglobulin G (IgG), hemoglobin, transferrin, human serum albumin, cytochrome c, and thrombin. Determination of cytochrome c and thrombin spiked into dilute serum samples showed no interference from the serum matrix. The benefit of porous properties of the affinity monolithic column was demonstrated by selective capture and preconcentration of thrombin at low ionic strength and subsequent rapid elution at high ionic strength. The combination of the polymer monolithic column and the aptamer affinities makes the aptamer-modified monolithic columns useful for protein detection and separation

Differentiation and Detection of PDGF Isomers and Their Receptors by Tunable Aptamer Capillary Electrophoresis

Tunable aptamer capillary electrophoresis (CE) techniques were developed to enable the separation and detection of platelet derived growth factor (PDGF) isomers and their receptors. Using an aptamer that formed a stable complex with the B chain but not with the A chain of PDGF, we were able to tweak the electrophoretic mobilities of the PDGF isomers for their separation. PDGF-AB bound to a single aptamer molecule was well resolved from PDGF-BB bound to two aptamer molecules. Simultaneous determination of 50 pM of two isomers was accomplished in a single analysis. Furthermore, PDGF-AB was used as a connector to bring receptor α and fluorescent aptamer into a single complex molecule. As a result, the formation of a (receptor α)-(PDGF-AB)-(fluorescent aptamer) ternary complex enabled the detection of the receptor α by tunable aptamer CE. A competitive assay was developed to determine receptor β, making use of the competition between the receptor β and fluorescent aptamer in binding to PDGF-BB. Detection limits were 0.5 nM for PDGF receptor α and 3 nM for receptor β. Determination of PDGF isomers and their receptors in diluted serum samples showed no interference from the sample matrix

Tunable Aptamer Capillary Electrophoresis and Its Application to Protein Analysis

A tunable aptamer electrophoretic assay enables highly sensitive fluorescence detection of multiple proteins and protein isomers. The electrophoretic mobility of proteins is tuned with DNA aptamers binding to the target proteins. Fluorescently labeled aptamers of varying nucleotide lengths serve as both charge modulators for capillary electrophoresis separation and as fluorescent affinity probes for ultrasensitive laser-induced fluorescence detection. Simultaneous determination of pM levels of human immunodeficiency virus reverse transcriptase, thrombin, human immunoglobulin E, and two isomers of platelet-derived growth factor in dilute human serum demonstrates the potential applications of this assay to biomarker development

Mass Spectrometry Identification of <i>N</i>‑Chlorinated Dipeptides in Drinking Water

We report the identification of <i>N</i>-chlorinated dipeptides as chlorination products in drinking water using complementary high-resolution quadrupole time-of-flight (QTOF) and quadrupole ion-trap mass spectrometry techniques. First, three model dipeptides, tyrosylglycine (Tyr-Gly), tyrosylalanine (Tyr-Ala), and phenylalanylglycine (Phe-Gly), reacted with sodium hypochlorite, and these reaction solutions were analyzed by QTOF. <i>N</i>-Cl-Tyr-Gly, <i>N</i>,<i>N</i>-di-Cl-Tyr-Gly, <i>N</i>-Cl-Phe-Gly, <i>N</i>,<i>N</i>-di-Cl-Phe-Gly, <i>N</i>-Cl-Tyr-Ala, and <i>N</i>,<i>N</i>-di-Cl-Tyr-Ala were identified as the major products based on accurate masses, ³⁵Cl/³⁷Cl isotopic patterns, and MS/MS spectra. These identified <i>N</i>-chlorinated dipeptides were synthesized and found to be stable in water over 10 days except <i>N</i>,<i>N</i>-di-Cl-Phe-Gly. To enable sensitive detection of <i>N</i>-chlorinated dipeptides in authentic water, we developed a high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method with multiple reaction monitoring (MRM) mode. <i>N</i>-Cl-Tyr-Gly, <i>N</i>,<i>N</i>-di-Cl-Tyr-Gly, <i>N</i>-Cl-Phe-Gly, <i>N</i>-Cl-Tyr-Ala, and <i>N</i>,<i>N</i>-di-Cl-Tyr-Ala along with their corresponding dipeptides were detected in authentic tap water samples. The dipeptides were clearly detected in the raw water, but the <i>N</i>-chlorinated dipeptides were at background levels. These results suggest that the <i>N</i>-chlorinated dipeptides are produced by chlorination. This study has identified <i>N</i>-chlorinated dipeptides as new disinfection byproducts in drinking water. The strategy developed in this study can be used to identify chlorination products of other peptides in drinking water

DNA Aptamers Binding to Multiple Prevalent M-Types of <i>Streptococcus pyogenes</i>

This paper describes the selection of high affinity DNA aptamers binding to multiple M-types of the pathogenic species Streptococcus pyogenes (Group A Streptococcus or GAS). Unlike common aptamer selection techniques that use purified molecules of a monoclonal cell population as targets, this work has achieved the selection of aptamers against the various M-types of S. pyogenes. Cell mixtures containing equal numbers of the 10 most prevalent S. pyogenes M-types were incubated with 80-nucleotide DNA libraries, centrifuged, and washed to separate cell-bound from unbound DNA sequences. The DNA bound to the cells was amplified using the polymerase chain reaction, and the amplicons were tested for their binding to the target cells. The amplicons were also used as new DNA libraries for subsequent rounds of selection. Cloning, sequencing, and subsequent analysis of selected aptamers showed that they bind preferentially to GAS over other common and related bacteria. Resultant DNA aptamers showed strong and preferential binding to GAS, including the 10 most prevalent GAS M-types and another 10 minor M-types tested. Estimated Kd values were in the range of 4 to 86 nM. Two aptamers, 20A24P and 15A3P (with estimated binding dissociation constants of 9 and 10 nM, respectively), are particularly promising. These aptamers could potentially be used to improve the detection of GAS, a pathogen that is the causative agent of many infectious diseases, most notably strep throat

Halobenzoquinone-Induced Alteration of Gene Expression Associated with Oxidative Stress Signaling Pathways

Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) that effectively induce reactive oxygen species and oxidative damage in vitro. However, the impacts of HBQs on oxidative-stress-related gene expression have not been investigated. In this study, we examined alterations in the expression of 44 genes related to oxidative-stress-induced signaling pathways in human uroepithelial cells (SV-HUC-1) upon exposure to six HBQs. The results show the structure-dependent effects of HBQs on the studied gene expression. After 2 h of exposure, the expression levels of 9 to 28 genes were altered, while after 8 h of exposure, the expression levels of 29 to 31 genes were altered. Four genes (<i>HMOX1</i>, <i>NQO1</i>, <i>PTGS2</i>, and <i>TXNRD1</i>) were significantly upregulated by all six HBQs at both exposure time points. Ingenuity pathway analysis revealed that the Nrf2 pathway was significantly responsive to HBQ exposure. Other canonical pathways responsive to HBQ exposure included GSH redox reductions, superoxide radical degradation, and xenobiotic metabolism signaling. This study has demonstrated that HBQs significantly alter the gene expression of oxidative-stress-related signaling pathways and contributes to the understanding of HBQ-DBP-associated toxicity

Aptamer Capturing of Enzymes on Magnetic Beads to Enhance Assay Specificity and Sensitivity

Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers

Aptamer-Based Affinity Chromatographic Assays for Thrombin

Affinity chromatographic assays for thrombin were developed using two aptamers as affinity ligands. The efficient capture and step elution of thrombin with NaClO4 enabled the determination of thrombin by using either absorbance or fluorescence detection. Preconcentration of thrombin on the affinity column improved the detection limit of thrombin to 0.1 nM. Using an aptamer for the fibrinogen-binding site of thrombin and a second aptamer for the heparin-binding site, a sandwich chromatographic assay was developed, showing improved selectivity of thrombin detection and eliminating the need for labeling thrombin in the sample. The increased local concentration of aptamers immobilized on monolithic columns favored the formation of aptamer−thrombin complexes, resulting in improved retention and detection of thrombin at trace levels

Quantification of Viable but Nonculturable <i>Escherichia coli</i> O157:H7 by Targeting the <i>rpoS</i> mRNA

Escherichia coli O157:H7 easily becomes viable but nonculturable (VBNC) under environmental stresses and escapes detection by current methods. Here, we report a unique method enabling the quantification of VBNC E. coli O157:H7 using a selective marker within the rpoS gene. A nucleotide at position +543 within the rpoS gene open reading frame was identified to be unique to E. coli O157:H7. Specifically designed primers and probe combinations were able to differentiate E. coli O157:H7 from closely related bacteria and other common bacteria. The application of this strategy correctly identified 36 clinical and bovine isolates of E. coli O157:H7. A one-step quantification method combining reverse transcription (RT) and real-time quantitative polymerase chain reaction (qPCR) was developed to provide a linear relationship (R2 > 0.99) of copies of RNA with threshold cycles (Ct) and the capability of detecting a single copy of rpoS RNA standards. This technique was used to determine the copies of the rpoS mRNA in culturable cells at different growth phases (mid-log, late-log, and stationary phase) to be 1.57, 0.56, and 0.41 copies/CFU, respectively. VBNC E. coli O157:H7 was determined to have one copy of the rpoS mRNA for every 10 cells, and no rpoS mRNA was detected in 106 dead cells and negative controls. This technique had a linear dynamic range over 6 orders of magnitude and >90% amplification efficiency for tap and river water samples. It was able to selectively quantify as few as 7 E. coli O157:H7 cells in pure culture, 9 culturable cells in tap water and river water, and 23 VBNC cells in river water, demonstrating the best quantification limits for culturable and VBNC E. coli O157:H7 in environmental water