Carbon nanotube decorated magnetic microspheres as an affinity matrix for biomolecules

Carbon nanotube (CNT) decorated magnetic microspheres were fabricated to develop a multimodal platform that utilizes non-covalent molecular interactions of CNTs to magnetically separate biomolecules. Hybrid CNT-microspheres prepared by a feasible method reported herein had a well-defined structure as characterized by Raman spectroscopy and scanning electron microscopy. Binding interactions of resulting magnetic CNT-microspheres with DNA oligonucleotides were studied to demonstrate that single stranded DNA (ssDNA) in a solution can be effectively recovered by magnetic CNT-microspheres through strong physical wrapping of DNA around CNTs' walls. The magnetic character of these CNT-microspheres combined with their capability to bind other molecules including DNA allows their use as an affinity matrix that can be utilized in affinity separation of biomolecules, and also as a platform to monitor non-covalent binding interactions of CNTs with other biomolecules. As a proof of concept, we report on the use of these CNT-microspheres in in vitro selection of ssDNA aptamers against carcinoembryonic antigen (CEA), a cancer biomarker, by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). ssDNA aptamer candidates that have strong affinity towards CEA were successfully separated magnetically from a pool of ssDNA (1014 molecules). Our results demonstrate that CNT-microspheres can serve as strong tools for affinity separation methodologies and can be utilized for various affinity pairs in solution

E. coli-quantum dot bioconjugates as whole-cell fluorescent reporters for probing cellular damage

A quantum dot (QD) conjugated whole-cell E. coli biosensor (E. coli–QD bioconjugates) was developed as a new molecular tool for probing cellular damage. The E. coli–QD bioconjugates were viable and exhibited fluorescence emission at 585 nm. Scanning electron microscopy (SEM) analysis of E. coli–QD bioconjugates revealed that the QDs were immobilized on the cell-surfaces and the fluorescence emission from QDs present on cell-surfaces was visualized by confocal microscopic examination. The E. coli–QD bioconjugates were employed as whole-cell fluorescent reporters that were designed to function as fluorescence switches that turn-off when cellular damage occurs. In this study, multi-walled carbon nanotubes (CNTs) were utilized as a model nanomaterial to probe cellular damage. Fluorescence spectra were recorded after the exposure of E. coli–QD bioconjugates with CNTs. We observed a strong correlation between fluorescence emission spectra, SEM and confocal microscopic analysis demonstrating that CNTs induced a dose and exposure time-dependent cellular toxicity. This toxicity mainly occurred by the physical interaction and cellular trafficking mechanisms that led to the collapse of the cellular structure and thus loss of fluorescence. The responses of E. coli–QD bioconjugates against CNTs were also visualized by simply exposing the cells to UV light and therefore rapid toxicity analysis and screening can be made. Our study demonstrated an easy and simple method to determine an important mechanistic perspective for the biological toxicity of chemicals or nanomaterials (NMs)

Biosensors for cardiac biomarkers detection: a review

The cardiovascular disease (CVD) is considered as a major threat to global health. Therefore, there is a growing demand for a range of portable, rapid and low cost biosensing devices for the detection of CVD. Biosensors can play an important role in the early diagnosis of CVD without having to rely on hospital visits where expensive and time-consuming laboratory tests are recommended. Over the last decade, many biosensors have been developed to detect a wide range of cardiac marker to reduce the costs for healthcare. One of the major challenges is to find a way of predicting the risk that an individual can suffer from CVD. There has been considerable interest in finding diagnostic and prognostic biomarkers that can be detected in blood and predict CVD risk. Of these, C-reactive protein (CRP) is the best known biomarker followed by cardiac troponin I or T (cTnI/T), myoglobin, lipoprotein-associated phospholipase A(2), interlukin-6 (IL-6), interlukin-1 (IL-1), low-density lipoprotein (LDL), myeloperoxidase (MPO) and tumor necrosis factor alpha (TNF-α) has been used to predict cardiovascular events. This review provides an overview of the available biosensor platforms for the detection of various CVD markers and considerations of future prospects for the technology are addressed

A new lab-on-chip transmitter for the detection of proteins using RNA aptamers

A new RNA aptamer based affinity biosensor for CReactive Protein (CRP), a risk marker for cardiovascular disease was developed using interdigitated capacitor (IDC), integrated in Voltage Controlled Oscillator (VCO) and output signal is amplified using Single Stage Power Amplifier (PA) for transmitting signal to receiver at Industrial, Scientific and Medical (ISM) band. The Lab-on-Chip transmitter design includes IDC, VCO and PA. The design was implemented in IHP 0.25μm SiGe BiCMOS process; post-CMOS process was utilized to increase the sensitivity of biosensor. The CRP was incubated between or on interdigitated electrodes and the changes in capacitance of IDC occurred. In blank measurements, the oscillation frequency was 2.464GHz whereas after RNA aptamers were immobilized on open aluminum areas of IDC and followed by binding reaction processed with 500pg/ml CRP solution, the capacitance shifted to 2.428GHz. Phase noise is changed from -114.3dBc/Hz to -116.5dBc/Hz

Sensitive detection of Nampt(PBEF/Visfatin)in human serum for point-of-care applications using aptamer based capacitive biosensor

NAMPT is a multifunctional protein, also known as visfatin or pre-B cell colony-enhancing factor, which exists as the rate-limiting intracellular enzyme for nicotinamide adenine dinucleotide (NAD) synthesis starting from nicotinamide [1]. The plasma Nampt levels are reported to have correlation with obesity and obese related metabolic disease, such as Type 2 diabetes mellitus (T2DM), cardiovascular diseases [2] and hyperlipidemia [3] due to association with lipoprotein and cholesterol. Therefore, sensitive detection of Nampt potentially enable accurate diagnosis of T2DM, cardiovascular and hyperlipidemia diseases. In this study, for the first time, we developed an ssDNA aptamer that specifically bind Nampt (Kd=72.52 nM) in human serum by systematic evolution of ligands by exponential enrichment (SELEX) process. Nampt-specific ssDNA aptamers were then applied as the recognition molecules for the development of a capacitive biosensor using non-Faradaic impedance spectroscopy (nFIES), which converts the biological binding event into a quantifiable signal for sensitive and efficient detection of the Nampt (Fig. 1). The interaction of aptamer-Nampt induced the change in dielectric properties, charge distribution, and conductivity. The limit of detection was 1 ng/ml with a dynamic range of upto 50 ng/ml in serum and this range is under the clinical requirements both in the normal Nampt levels, which is 15.8 ng/ml, and in the T2DM patients level, which is 31.9 ng/ml. This assay system for Nampt detection using aptamers is a potential alternative approach for applications in clinical studies and Point-Of-Care health technologies

Revealing the molecular interactions of aptamers that specifically bind to the extracellular domain of HER2 cancer biomarker protein: An in silico assessment

Single-stranded (ss) oligonucleotide aptamers are emerging as the promising substitutes for monoclonal antibodies because of their low production cost and good batch-to-batch consistency. Aptamers vividly bind to a variety of cellular targets and alter their functions with a remarkable degree of specificities. In this study, the positive clones of human epidermal growth factor receptor 2 (HER2) specific binding ssDNA aptamers which were previously identified by in vitro Systematic Evolution of Ligands by EXponential enrichment (SELEX) process, hitherto lacking the putative binding site information and residues crucial for aptamer recognition are studied. Primarily, four putative DNA binding regions present on the HER2 extracellular domain (ECD) were identified using prediction servers and electrostatic potential maps, which were further exploited to delineate the aptamer binding features. Molecular docking and molecular dynamics (MD) simulations revealed stable binding nature of three aptamers (H2>H1>H6), which chose Site 2a as preferred binding site present on the HER2(ECD) protein. Furthermore, amino acid residues viz. Asn37, Gln59, Arg81-Gln84, Asp88, and Lys128 of Site 2a were found to be crucial for high-affinity binding. This knowledge can be utilized as a benchmark for the future studies, in search for better and highly specific anti-HER2 aptamers as cancer therapeutics or as diagnostic agents

Label-free capacitance based aptasensor platform for the detection of HER2/ErbB2 cancer biomarker in serum

In this study, a label-free capacitive aptasensor was developed based on capturing of human epidermal growth factor receptor 2 (HER2) protein by anti-HER2 ssDNA aptamers functionalized on interdigitated microelectrodes of capacitor as bio-recognition elements. The aptasensor response was measured by non-Faradaic Impedance Spectroscopy (nFIS) method. Capacitance signal specific to target HER2 protein is based on changes occurred due to charge distribution upon interaction of aptamer-protein molecules against to the applied AC frequency (50-350 MHz). HER2 protein in spiked human serum was successfully detected through concentration dependent changes in impedance/capacitance values as a result of the formation of aptamer-HER2 protein complex (0.2-2 ng mL(-1) of HER2) on capacitor microelectrodes. The label-free capacitive aptasensing is a versatile and promising approach for early detection of cancer biomarkers in dilute human serum and the method potentially be extended to a wide variety of diseases biomarkers