Analytical applications of aptamers Tombelli, S.; Minunni, M.; Mascini, M.
Biosensors & bioelectronics,
06/2005, Letnik:
20, Številka:
12
Journal Article
Recenzirano
So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, ...selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10,000
Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets.
Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.
Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods, which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process.
Fluorescence imaging coupled with nanotechnology is making possible the development of powerful tools in the biological field for applications such as cellular imaging and intracellular messenger RNA ...monitoring and detection. The delivery of fluorescent probes into cells and tissues is currently receiving growing interest because such molecules, often coupled to nanodimensional materials, can conveniently allow the preparation of small tools to spy on cellular mechanisms with high specificity and sensitivity. The purpose of this review is to provide an exhaustive overview of current research in oligonucleotide optical switches for intracellular sensing with a focus on the engineering methods adopted for these oligonucleotides and the more recent and fascinating techniques for their internalization into living cells. Oligonucleotide optical switches can be defined as specifically designed short nucleic acid molecules capable of turning on or modifying their light emission on molecular interaction with well-defined molecular targets. Molecular beacons, aptamer beacons, hybrid molecular probes, and simpler linear oligonucleotide switches are the most promising optical nanosensors proposed in recent years. The intracellular targets which have been considered for sensing are a plethora of messenger-RNA-expressing cellular proteins and enzymes, or, directly, proteins or small molecules in the case of sensing through aptamer-based switches. Engineering methods, including modification of the oligonucleotide itself with locked nucleic acids, peptide nucleic acids, or
l
-DNA nucleotides, have been proposed to enhance the stability of nucleases and to prevent false-negative and high background optical signals. Conventional delivery techniques are treated here together with more innovative methods based on the coupling of the switches with nano-objects.
Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the ...thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads.
The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430
nM for thrombin was achieved. A lower detection limit for the protein (175
nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45
nM of thrombin demonstrating the best analytical performances.
With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.
The in vitro selection of combinatorial libraries of RNA/DNA, has allowed the identification of specific nucleic acids (aptamers) which bind to a wide range of target molecules with high affinity and ...specificity.
In this work, an RNA aptamer, specific for the protein trans-activator of transcription (Tat) of HIV-1, has been used as bio-recognition element to develop a biosensor (aptasensor). The biosensor was optimised using piezoelectric quartz-crystals as transducers and the aptamer was immobilised on the gold electrode of the crystal. The immobilisation procedure was based on the interaction between the biotinylated aptamer and streptavidin previously deposited on the electrode.
The main analytical characteristics of the biosensor, such as sensitivity, selectivity and reproducibility, have been studied in details. An optimised regeneration procedure allowed the multiple use of the aptamer-coated crystal.
The aptasensor has been compared with the corresponding immunosensor, based on the specific monoclonal anti-Tat antibody. The antibody was immobilised on a layer of carboxylated dextran previously deposited on the gold electrode.
The results demonstrated that the use of a biosensor with a specific aptamer as bio-recognition element could be an interesting approach in the detection of proteins, which has been here examined considering a model system.
Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an ...iterative process of adsorption, recovery and amplification. This review described the
in vitro process to obtain aptamers (SELEX). It mentions the main characteristics of these molecules (i.e. affinity, specificity and stability). Moreover, it discusses advantages over antibodies. It reports potential applications of aptamers in analytical and diagnostic assays as biocomponents of biosensors (aptasensors) and allosteric ribozymes (aptazymes).
The development of a RNA-aptamer-based optical biosensor (aptasensor) for C-reactive protein (CRP) is reported. CRP is an important clinical biomarker; it was the first acute-phase protein to be ...discovered (1930) and is a sensitive systemic marker of inflammation and tissue damage. It has also a prognostic value for patients with acute coronary syndrome. The average concentration of CRP in serum is 0.8 ppm and it increases in response to a variety of inflammatory stimuli, such as trauma, tissue necrosis, infection and myocardial infarction. The interaction between the 44-base RNA aptamer and the target analyte CRP is studied. In particular, the influence of the aptamer immobilization procedure (chemistry, length, concentration), as well as the binding conditions, i.e., the influence on the binding of different buffers, the presence of Ca²⁺ ion and the specificity (against human serum albumin) have been evaluated. Using the best working conditions, we achieved a detection limit of 0.005 ppm, with good selectivity towards human serum albumin. Some preliminary experiments in serum are reported. graphic removed
In this paper, a simple and sensitive approach for human epidermal growth factor receptor 2 (HER2) detection is presented, using antibody‐functionalised magnetic beads coupled to screen‐printed ...cells. The immunoassay is based on a sandwich format in which a primary monoclonal antibody anti‐HER2 is coupled to protein A modified magnetic beads. The modified beads are then used to capture the protein from the sample solution and a sandwich assay is performed by adding a secondary monoclonal antibody anti‐HER2 labelled with biotin. The enzyme alkaline phosphatase (AP) conjugated with streptavidin and its substrate (1‐naphthyl‐phosphate) are then used for the electrochemical detection by differential pulse voltammetry (DPV). The experimental conditions for the immunoassay were optimised. The performance of the assay in terms of sensitivity, reproducibility and selectivity has been studied in buffer and serum samples from hospital patients.
Here we present a new generic opto-bio-sensing platform combining immobilised aptamers on an infrared plasmonic sensing device generated by nano-structured thin film that demonstrates amongst the ...highest index spectral sensitivities of any optical fibre sensor yielding on average 3.4 × 10
nm/RIU in the aqueous index regime (with a figure of merit of 330) This offers a single stage, solution phase, atto-molar detection capability, whilst delivering real-time data for kinetic studies in water-based chemistry. The sensing platform is based upon optical fibre and has the potential to be multiplexed and used in remote sensing applications. As an example of the highly versatile capabilities of aptamer based detection using our platform, purified thrombin is detected down to 50 attomolar concentration using a volume of 1mm
of solution without the use of any form of enhancement technique. Moreover, the device can detect nanomolar levels of thrombin in a flow cell, in the presence of 4.5% w/v albumin solution. These results are important, covering all concentrations in the human thrombin generation curve, including the problematic initial phase. Finally, selectivity is confirmed using complementary and non-complementary DNA sequences that yield performances similar to those obtained with thrombin.
The high sensitivity and specificity of DNA hybridisation techniques makes them powerful tools for environmental or clinical analysis. This work describes the development of a DNA piezoelectric ...biosensor for the detection of the hybridisation reaction. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of oligonucleotides onto the gold surface of the quartz crystal. Four immobilisation procedures were tested and compared considering the amount of immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non-specific adsorption. All the experiments were performed with oligonucleotides of 25 bases (probe, target and non-complementary oligonucleotide). The four coating methods were all based on the use of self-assembled monolayers (SAM). Three of them employed the interaction between streptavidin and biotin for the immobilisation of a biotinylated probe. Results indicated that immobilisation of a biotinylated probe on streptavidin linked to a layer of carboxylated dextran provides higher sensitivity for the detection of the hybridisation reaction, absence of non-specific adsorption and a higher stability with respect to the regeneration step.
Three different biosensors for detection of Genetically Modified Organisms (GMOs) are presented. The sensing principle is based on the affinity interaction between nucleic acids: the probe is ...immobilised on the sensor surface and the target analyte is free in solution. The immobilised probes are specific for most inserted sequences in GMOs: the promoter P35S and the terminator TNOS. Electrochemical methods with screen-printed electrodes, piezoelectric and optical (SPR) transduction principles were applied.
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