Immobilization Of Biosensors Research Articles

Hydrogel-Embedded Quantum Dot-Transcription Factor Sensors for Quantitative Progesterone Detection.

Immobilization of biosensors in or on a functional material is critical for subsequent device development and translation to wearable technology. Here, we present the development and assessment of an immobilized quantum dot-transcription factor-nucleic acid complex for progesterone detection as a first step toward such device integration. The sensor, composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA, is embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot-transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot-transcription factor DNA assembly resulting in an attenuated ratiometric fluorescence output via Förster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeated analyte measurements are similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor-DNA assembly as a recognition/transduction element, offering a promising framework for future biosensor devices.

Surface Immobilized Nucleic Acid-Transcription Factor Quantum Dots for Biosensing.

Immobilization of biosensors on surfaces is a key step toward development of devices for real-world applications. Here the preparation, characterization, and evaluation of a surface-bound transcription factor-nucleic acid complex for analyte detection as an alternative to conventional systems employing aptamers or antibodies are described. The sensor consists of a gold surface modified with thiolated Cy5 fluorophore-labeled DNA and an allosteric transcription factor (TetR) linked to a quantum dot (QD). Upon addition of anhydrotetracycline (aTc)-the analyte-the TetR-QDs release from the surface-bound DNA, resulting in loss of the Förster resonance energy transfer signal. The sensor responds in a dose-dependent manner over the relevant range of 0-200 µm aTc with a limit of detection of 80 nm. The fabrication of the sensor and the subsequent real-time quantitative measurements establish a framework for the design of future surface-bound, affinity-based biosensors using allosteric transcription factors for molecular recognition.

Stable immobilisation of His-tagged proteins on BLI biosensor surface using cobalt

In biosensor experiments, ligand molecules need to be immobilized tightly on sensor surfaces for sensitive and robust detection of the analyte of interest. Stretches of histidines (His-tags) are typically inserted into recombinant proteins for affinity purification purposes, but can also be used in the immobilization of biosensors functionalized with Ni-NTA (nitrilotriacetic acid). The His-tag-Ni-NTA bond is, however, easily disrupted by factors including low pH, reducing agents, or chelating agents such as EDTA. In biosensing applications, a stable ligand immobilization that tolerates harsh conditions is preferable, because the sample matrix can vary from bodily fluids to waste waters. We describe here Co(III)-NTA bonding for tight immobilization of His-tagged proteins on Octet BLI-biosensors. The performance of the Ni(II)-NTA was compared to that of Co(III)-NTA using His-tagged avidins and norovirus proteins. Comparative studies were performed using biotin-iminodiacetic acid (IDA) for streptavidin sensor functionalization instead of the NTA surfaces provided by the manufacturer. It was noted that Co(III)-NTA offers highly stable immobilization of His-tagged protein, tolerating factors such as 0.7M imidazole, that is typically used for detaching proteins from Ni(II)-NTA-agarose used in protein purification columns. The good performance of Co(III)-NTA functionalized BLI sensor was demonstrated by detecting norovirus antibodies from human serum samples with sensors functionalized with His-tagged virus-like particles. The use of Co-NTA sensors for attachment of His-tagged proteins is ideal in conditions, where the immobilized ligands need to be firmly attached and tolerate harsh chemical conditions.

Immobilization of fluorescent whole cell biosensors for the improved detection of heavy metal pollutants present in aquatic environment

Fluorescent whole cell bacterial biosensors are very specific in detection of toxic heavy metal pollutants present in environmental water samples. However, detection of the actual fluorescence intensity emitted by the biosensors is a technological challenge because the biosensors remain in suspended form and there can be no control over the cell movement and multiplication. Immobilization of these biosensors is essential to develop a more stable, robust and sustainable detection system that is able to measure the actual intensity. In this report, we have established Polyethylenimine (PEI), a synthetic polymer, as the promising immobilizing agent for three fluorescent whole cell Escherichia coli DH5α biosensors which detect Hg(II), Pb(II) and Zn(II) respectively within a specified range. These recombinant biosensors harbor a gene for reporter protein called GFP (Green Fluorescent Protein), whose heavy metal induced expression leads to fluorescence which is directly proportional to the concentration of the heavy metals present in the water samples. For immobilization, the log phase cultures of Hg(II), Pb(II) and Zn(II) biosensors were adsorbed on the respective semi-dried PEI matrix fixed on the glass slides. Each of the three immobilized biosensors remained viable up to 24 h without being fed with any nutrients. Thus, the immobilization efficiency of PEI in terms of sustaining the viable biosensors with stable fluorescence was found to be 86.21%, 90.11% and 87.32% for Hg(II), Pb(II) and Zn(II) biosensors respectively. Whereas, without immobilization the biosensors could retain viability and fluorescence ability for only 4 hours. Hence, PEI provides a suitable environment for the stable sustenance of all the three biosensors along with enhanced target selectivity and consistent sensitivity in detection of Hg(II), Pb(II) and Zn(II) pollutants present in environmental water samples.

Preparation of graphene nanoplatelet–titanate nanotube composite and its advantages over the two single components as biosensor immobilization materials

A novel nanocomposite consisting of graphene nanoplatelets (GNPs) and titanate nanotubes (TNTs) have been synthesized successfully utilizing the hydrothermal method. The GNP–TNT composite was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and electrochemical impedance spectroscopy. The voltammetric characterization of GNP–TNT composite, pure GNPs and pure TNTs modified horseradish peroxidase (HRP) biosensors were conducted to select the most suitable electrode immobilization material for enzyme biosensors. The GNPs was firstly eliminated owing to its extremely high background charging current, distinct electrochemical interference from its surface functional groups and low signal to noise ratio. Next, the direct electron transfer of HRP on electrode and the catalytic current of HRP towards H2O2 was increased around 45% and 72% respectively on GNP–TNT composite modified electrodes compared with those on pure TNTs modified electrodes. GNP–TNT composite modified HRP biosensor also exhibited superiority over pure TNTs modified HRP biosensor in the analytical performance. The precision and stability study provided additional evidence for the feasibility of using GNP–TNT composite as electrode modification material.

Electrochemical Biosensor Immobilization of Formaldehyde Dehydrogenase with Nafion for Determination of Formaldehyde from Indian Mackerel (Rastrelliger kanagurta) Fish

An electrochemical biosensor was developed based on formaldehyde dehydrogenase immobilized with Nafion membrane for determination of formaldehyde in fish. The enzyme was immobilized through the entrapment technique and measured based on the reduction of I²-nicotinamide adenine dinucleotide. The response time of the formaldehyde biosensor was <1 min, with an optimum pH of 8. The optimum enzyme loading and NAD + concentrations were found at 30 mg/mL and 0.5 mM, respectively. Using the formaldehyde biosensor, a linear response of formaldehyde showed a range of 0.1 to 10 ppm and a detection limit of 0.016 ppm. In application of Nash method, the samples were stored at 4°C ± 1 for 10 days. With the two combined methods, a linear correlation coefficient with R ² = 0.9982 (y = 0.956x - 0.014) was found. The developed formaldehyde biosensor showed a good reproducibility, long storage stability (more than 6 months stored at 4°C), and also effective monitoring of formaldehyde level in Indian mackerel (Rastrelliger kanagurta) fish.

An automated peptide and protein thiazolidine coupling chemistry for biosensor immobilization giving a unique N-terminal orientation

1 Abbreviations used: Pms, 2-(phenyl(methyl)sulfonio)ethyloxycarbonyl; mAb, monoclonal antibody; EGFR, epidermal growth factor receptor; Peptide and protein sensor chips and microarrays are important tools for biorecognition analysis for the study of both the functions and interactions of biomolecules [1– 4]. They can also be employed in high-throughput assays that require only minute amounts of analyte [5–7]. The success of such studies is dependent on the preparation of appropriately derivatized surfaces. The attachment of peptides and proteins in a specific orientation rather than in a random manner can result in a substantial increase in assay sensitivity and selectivity [1,8]. A number of methods to achieve this have been reported previously. Proteins and peptides containing single free sulfhydryl groups have been immobilized in a defined orientation by thiol capture [1,4,9–11]. A traceless version of the Staudinger ligation, in which an azido-peptide or azido-protein is reacted with a phosphinothioester-derived slide to generate a functional peptide microarray, has been used [12]. We have previously described a simple means of immobilizing N-terminal serineand threonine-containing peptides and proteins onto surfaces bearing streptavidin [13]. Derivatization of the peptide or protein following conversion into an N-terminal carbonyl precursor was achieved by covalent attachment of a water-soluble dipeptide containing a lysine residue bearing an N-cysteine-derived 1,2-aminothiol and an N-biotin moiety. However, each of these immobilization strategies has both advantages and disadvantages, with the latter including the need for a conveniently located functional group or amino acid within the peptide and protein to enable subsequent immobilization in an orientation that will allow