Javed H. Niazi

ssDNA Aptamer-Based Surface Plasmon Resonance Biosensor for the Detection of Retinol Binding Protein 4 for the Early Diagnosis of Type 2 Diabetes

Retinol binding protein 4 (RBP4) is a useful biomarker in the diagnosis of type 2 diabetes since its level in the serum is higher in insulin-resistant states. Accurate measurement of the serum RBP4 levels is hampered by conventional immunologic methods, such as enzyme-linked immunosorbent assay (ELISA). In this study, therefore, we have developed an aptamer-based surface plasmon resonance (SPR) biosensor that can be used to sense for RBP4 in serum samples. A single-stranded DNA (ssDNA) aptamer that showed high affinity (Kd = 0.2 +/- 0.03 microM) and specificity to RBP4 was selected. This RBP4-specific aptamer was immobilized on a gold chip and used in a label-free RBP4 detection using SPR. Analysis of RBP4 in artificial serum using SPR was compared with ELISA and Western blot analysis. Our results indicated that the RBP4-specific aptamer-based SPR biosensor gave better dose-dependent responses and was more sensitive than ELISA assays. As such, this RBP4 aptamer-based SPR biosensor can be potentially used to monitor the RBP4 levels within the serum as an indicator of type 2 diabetes.

Single-stranded DNA aptamers specific for antibiotics tetracyclines

Tetracyclines (TCs) are a group of antibiotics comprising of a common tetracycline (TET) nucleus with variable X(1) and X(2) positions on 5 and 6 carbon atoms, such as oxytetracycline (OTC) and doxycycline (DOX). In this study, the tetracycline group specific (TGS) ssDNA aptamers were identified by modified SELEX method by employing tosylactivated magnetic beads (TMB) coated with OTC, TET, and DOX, respectively, as targets and counter targets. Twenty TGS-aptamers were selected, of which seven aptamers, designated as T7, T15, T19, T20, T22, T23, and T24, showed high affinity to the basic TET backbone (K(d)=63-483 nM). The specificity of these TGS-aptamers to structural analogues followed the order in which the TCs was employed during SELEX process (OTC>TET>DOX) except aptamer T22, which was highly specific to TET than OTC or DOX. Aptamers that were specific to one target molecule but fail to bind the other structurally related TCs were eliminated during counter selection steps. Three aptamers, T7, T19, and T23 contained palindromic consensus sequence motif GGTGTGG. The remaining TGS-aptamers showed many consensus sequences that are truncated forms of this palindrome forming mirror image or inverted sequences. For example, GTGG or its inverted form, GGTG motif was found in all TGS-aptamers. A consensus sequence motif TGTGCT or its truncated terminal T-residue was found in most TGS-aptamers, which is predicted to be essential for high affinity and group specificity. These TGS-aptamers have potential applications such as target drug delivery, and detection of TCs in pharmaceutical preparations and contaminated food products.

Specific detection of oxytetracycline using DNA aptamer-immobilized interdigitated array electrode chip.

An electrochemical sensing system for oxytetracycline (OTC) detection was developed using ssDNA aptamer immobilized on gold interdigitated array (IDA) electrode chip. A highly specific ssDNA aptamer that bind to OTC with high affinity was employed to discriminate other tetracyclines (TCs), such as doxycycline (DOX) and tetracycline (TET). The immobilized thiol-modified aptamer on gold electrode chip served as a biorecognition element for the target molecules and the electrochemical signals generated from interactions between the aptamers and the target molecules was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV). The current decrease due to the interference of bound OTC, DOX or TET was analyzed with the electron flow produced by a redox reaction between ferro- and ferricyanide. The specificity of developed EC-biosensor for OTC was highly distinguishable from the structurally similar antibiotics (DOX and TET). The dynamic range was determined to be 1-100 nM of OTC concentration in semi-logarithmic coordinates.

Label-free capacitive biosensor for sensitive detection of multiple biomarkers using gold interdigitated capacitor arrays

In this study, a highly sensitive and label-free multianalyte capacitive immunosensor was developed based on gold interdigitated electrodes (GID) capacitor arrays to detect a panel of disease biomarkers. C-reactive protein (CRP), TNFalpha, and IL6 have strong and consistent relationships between markers of inflammation and future cardiovascular risk (CVR) events. Early detection of a panel of biomarkers for a disease could enable accurate prediction of a disease risk. The detection of protein biomarkers was based on relative change in capacitive/dielectric properties. Two different lab-on-a-chip formats were employed for multiple biomarker detection on GID-capacitors. In format I, capacitor arrays were immobilized with pure forms of anti-CRP, -TNFalpha, and -IL6 antibodies in which each capacitor array contained a different immobilized antibody. Here, the CRP and IL6 were detected in the range 25 pg/ml to 25 ng/ml and 25 pg/ml to 1 ng/ml for TNFalpha in format I. Sensitive detection was achieved with chips co-immobilized (diluted) with equimolar mixtures of anti-CRP, -IL6, and -TNFalpha antibodies (format II) in which all capacitors in an array were identical and tested for biomarkers with sequential incubation. The resulting response to CRP, IL6, and TNFalpha in format II for all biomarkers was found to be within 25 pg/ml to 25 ng/ml range. The capacitive biosensor for panels of inflammation and CVR markers show significant clinical value and provide great potential for detection of biomarker panel in suspected subjects for early diagnosis.

Global Gene Response in Saccharomyces cerevisiae Exposed to Silver Nanoparticles

Silver nanoparticles (AgNPs), exhibiting a broad size range and morphologies with highly reactive facets, which are widely applicable in real-life but not fully verified for biosafety and ecotoxicity, were subjected to report transcriptome profile in yeast Saccharomyces cerevisiae. A large number of genes accounted for ∼3% and ∼5% of the genome affected by AgNPs and Ag-ions, respectively. Principal component and cluster analysis suggest that the different physical forms of Ag were the major cause in differential expression profile. Among 90 genes affected by both AgNPs and Ag-ions, metalloprotein mediating high resistance to copper (CUP1-1 and CUP1-2) were strongly induced by AgNPs (∼45-folds) and Ag-ions (∼22-folds), respectively. A total of 17 genes, responsive to chemical stimuli, stress, and transport processes, were differentially induced by AgNPs. The differential expression was also seen with Ag-ions that affected 73 up- and 161 down-regulating genes, and most of these were involved in ion transport and homeostasis. This study provides new information on the knowledge for impact of nanoparticles on living microorganisms that can be extended to other nanoparticles.

ssDNA aptamers that recognize diclofenac and 2-anilinophenylacetic acid

A series of 56 ssDNA aptamer variants that bind to diclofenac (DCF) were selected from an initial pool of 2.4x10(14) ssDNA molecules by Flu-Mag SELEX process. Sequence analysis of these aptamer variants showed three major groups based on sequence similarity in their random N40 sequences. Out of these, four aptamers designated as D10/DA24, D22, D16, and D3 showed high affinity to DCF with K(d) values 100.64, 166.34, 148.73, and 42.7 nM, respectively. Secondary structures of these aptamers showed highly distinct features with typical stem and loop structures. Specificity tests with these four aptamer variants showed that D3 aptamer had higher specificity to DCF followed by 2-anilinophenylacetic acid (2APA), a structural analog of DCF. Whereas aptamers D16 and D22 showed higher specificity to 2APA compared to DCF as target used during selection process. Further, the D10/DA24 aptamer showed high affinity but no specificity to DCF. The DCF aptamers selected can be potential candidates for drug-delivery systems, specific detection of DCF and its derivatives in pharmaceutical preparations and contaminants.

Rapid and sensitive detection of Nampt (PBEF/visfatin) in human serum using an ssDNA aptamer-based capacitive biosensor

A single-stranded DNA (ssDNA) aptamer was successfully developed to specifically bind to nicotinamide phosphoribosyl transferase (Nampt) through systematic evolution of ligands by exponential enrichment (SELEX) and successfully implemented in a gold-interdigitated (GID) capacitor-based biosensor. Surface plasmon resonance (SPR) analysis of the aptamer revealed high specificity and affinity (K(d)=72.52 nM). Changes in surface capacitance/charge distribution or dielectric properties in the response of the GID capacitor surface covalently coupled to the aptamers in response to changes in applied AC frequency were measured as a sensing signal based on a specific interaction between the aptamers and Nampt. The limit of detection for Nampt was 1 ng/ml with a dynamic serum detection range of up to 50 ng/ml; this range includes the clinical requirement for both normal Nampt level, which is 15.8 ng/ml, and Nampt level in type 2 diabetes mellitus (T2DM) patients, which is 31.9 ng/ml. Additionally, the binding kinetics of aptamer-Nampt interactions on the capacitor surface showed that strong binding occurred with increasing frequency (range, 700 MHz-1 GHz) and that the dissociation constant of the aptamer under the applied frequency was improved 120-240 times (K(d)=0.3-0.6 nM) independent on frequency. This assay system is an alternative approach for clinical detection of Nampt with improved specificity and affinity.

An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays.

An aptamer can specifically bind to its target molecule, or hybridize with its complementary strand. A target bound aptamer complex has difficulty to hybridize with its complementary strand. It is possible to determine the concentration of target based on affinity separation system for the protein detection. Here, we exploited this property using C-reactive protein (CRP) specific RNA aptamers as probes that were immobilized by physical adsorption on carbon nanotubes (CNTs) activated gold interdigitated electrodes of capacitors. The selective binding ability of RNA aptamer with its target molecule was determined by change in capacitance after allowing competitive binding with CRP and complementary RNA (cRNA) strands in pure form and co-mixtures (CRP:cRNA=0:1, 1:0, 1:1, 1:2 and 2:1). The sensor showed significant capacitance change with pure forms of CRP/cRNA while responses reduced considerably in presence of CRP:cRNA in co-mixtures (1:1 and 1:2) because of the binding competition. At a critical CRP:cRNA ratio of 2:1, the capacitance response was dramatically lost because of the dissociation of adsorbed aptamers from the sensor surface to bind when excess CRP. Binding assays showed that the immobilized aptamers had strong affinity for cRNA (K(d)=1.98 μM) and CRP molecules (K(d)=2.4 μM) in pure forms, but low affinity for CRP:cRNA ratio of 2:1 (K(d)=8.58 μM). The dynamic detection range for CRP was determined to be 1-8 μM (0.58-4.6 μg/capacitor). The approach described in this study is a sensitive label-free method to detect proteins based on affinity separation of target molecules that can potentially be used for probing molecular interactions.

Toxicity of Metallic Nanoparticles in Microorganisms- a Review

Recent advances in the synthesis and development of nanoparticles (NPs) for wide applications has lead to a serious threat to both human and environmental health. NPs are highly reactive and catalytic in nature compared to their ions or bulk counterparts and thus applicable in various fields including drug delivery, electronics, optics, and therapeutics. Due to these applications, many varieties of NPs in massive amounts are being industrially produced. These NPs are discharged in to the environment and thus providing a path to enter into food chain via microorganisms and eventually disturbs the ecological balance. The NPs exhibit toxicity to living organisms mainly because of their small size (>100 nm), large surface-to-volume ratio and highly reactive facets. The microorganisms including bacteria present in the natural ecosystem are the primary targets that get exposed to NPs. Before these NPs enter into the food chain, it is imperative to evaluate the toxicity associated with NPs in microorganisms. The most convenient and rapid way is to perform toxicity analysis using microorganisms such as bacteria. Toxicity of nanomaterials using microorganisms such as E.coli, Pseudomonas, Bacillus as models for prokaryotes gives an insight into the toxic impacts of NPs. Toxicities associated with NPs in microorganisms is mainly related to their nano-size that cause membrane disorganization, generation of reactive oxygen species (ROS) and in some ases, oxidative DNA damage. In this review article we describe the toxicity of various nanoparticles in bacteria and provide a rationale for assessing nanotoxicity and discuss the current status on toxicity impacts on microorganisms.

A novel bioluminescent bacterial biosensor using the highly specific oxidative stress-inducible pgi gene

A new oxidative stress-responsive bacterial biosensor was constructed using the promoter of the pgi gene fused to the luxCDABE reporter. This strain (PGRFM) responded in a dose-dependent manner to methyl viologen (MV), a model redox chemical that results in oxidative stress. The responses of strain PGRFM to redox chemicals was strongly dependent on the available carbon source. For example, when the strain was grown under nutrient-limited conditions in the presence of glucose or gluconate it was capable of responding to low MV concentrations (0.6-19.3ppm), whereas the same cells grown in LB (a nutrient rich media) only responded to higher concentrations (4.9-625ppm). This allowed us to select PGRFMs growth conditions and extend the range of concentrations at which a stress-inducing chemical could be detected. Further, strain PGRFM responded to structural analogs of MV (i.e., ethyl and benzyl viologen), demonstrating that this strain is responsive to the presence of superoxide radicals, regardless of the chemical by which they are generated. Strain PGRFMs response patterns to these analogs were distinct from each other, which determined their strength to induce oxidative stress. As well, a significant induction was seen when this strain was exposed to hydrogen peroxide, illustrating that strain PGRFM is responsive in the presence of both the superoxide (O(2)(-)) and hydroxyl (OH) radicals.