There is a high demand for convenient methodologies for observing and measuring the levels of specific proteins in biological and environmental samples because their detection, identification, and quantification can be very complex, expensive, and time-consuming. Biosensors are interesting tools offering certain operational advantages over standard photometric methods, notably with regard to celerity, ease-of-use, cost, simplicity, portability, and ease of mass production.
Biosensors have been developed for more than 25 years now, and have been commercialized for some particular applications like blood glucose and lactate measurement or bioprocess control, amongst others. However, they have not entered the market as much as expected, which is caused by several reasons. One reason is the instability of the biological recognition component of the biosensor (e.g. enzymes, cells, or antibodies).
Aptamers, which are ssDNA or RNA oligonucleotides, can adhere to their targets due to their specific three-dimensional structures; they offer specific properties which favor them as new biorecognition elements for biosensors. In particular, their outstanding and modifiable stability and their regenerative target binding assure the development of a new biosensor generation.
Although aptamers have been developed for all categories of targets ranging from small molecules to large proteins and even cells, proteins seem to be the biggest group of target molecules. In principle, it should be possible to produce aptamers for virtually every protein target. However, it is striking that there is only a small range of proteins that are detected using aptasensors.
Biosensor
As per the definition of IUPAC, a biosensor is an integrated receptor-transducer device, which is capable of providing selective quantitative or semi-quantitative analytical information. The biosensor consists, on the one hand, of a biological recognition component, which acts upon a biochemical mechanism, and, on the other hand, of a transducer relying on electrochemical, mass, optical, or thermal principles.
The characteristic trait of a biosensor is the direct spatial contact between the biological recognition component (or bioreceptor) and the transducing component. Typical bioreceptors in biosensors are enzymes, antibodies, microorganisms, and nucleic acids. Aptamers are a new promising group of bioreceptors because of their outstanding selectivity, sensitivity, and stability, the reproducibility of the target binding reaction, their production by chemical synthesis ensuring a constant lot-to-lot quality, and the ease of regeneration of aptamer-derived surfaces.
Biosensor principle
A biosensor consists of a bioreceptor for the specific sensing of the several analyte in spatial contact to a transducer for converting the signal into an electrically manageable format and a signal processing unit.
Protein biosensor sensing rules based on aptamers
Biosensors for protein sensing mainly involve antibodies, but recently, also aptamers have been used as biological recognition elements in the case of specific sensing, and enzymes in the case of entire protein sensing. Aptamers can match antibodies in a number of applications. Aptamers are very small in size (ca. 30 to 100 bases) in comparison to other biorecognition molecules like antibodies or enzymes. This allows efficient immobilization at high density.
Therefore, production, miniaturization, integration, and automation of biosensors can be accomplished more easily with aptamers than with antibodies. Once selected, aptamers can be synthesized with high reproducibility and purity. DNA aptamers are usually highly chemically stable, enabling reuse of the biosensors. In contrast, RNA aptamers are susceptible to degradation by the endogenous ribonucleases typically found in cell lysates and serum.
Therefore, biosensors utilizing RNA aptamers as bio-recognition elements can be used only for single-shot measurements in biological milieus. In order to overcome this problem, modifications of the 2′ positions of pyrimidine bases with amino/fluoro groups have been introduced. Another possibility is the use of RNase inhibitors. The significant conformational change of most aptamers upon target binding offers great flexibility in the design of biosensors with high sensing sensitivity and selectivity.
Protein targets, with their high structural complexity, allow aptamer binding by stacking interactions, shape complementarity, electrostatic interactions, and hydrogen bonding. Furthermore, in theory, proteins can present more than one binding site for aptamers, allowing the selection of a pair of aptamers binding to different parts of the target and enabling sandwich-assay-based biosensors.
Electrochemical aptasensors
Electrochemical transduction of biosensors using aptamers as bioreceptors includes methods like Faradaic Impedance Spectroscopy (FIS), differential pulse voltammetry, square wave voltammetry, potentiometry, or amperometry. In theory, it can be differentiated between a positive or negative readout signal, i.e. an increase or a decrease of response following receptor-target interaction.
Xu et al. demonstrated an electrochemical impedance spectroscopy sensing method for aptamer-modified array electrodes as a promising label-free sensing method for IgE. They compared DNA aptamer-based electrodes with anti-human IgE antibody-based electrodes and found lower background noise, decreased nonspecific surface adsorption, and larger differences in the impedance signals due to the small size and simple construction of the aptamers in comparison to the antibody.
Electric resistance detectors allow real-time monitoring of the sensor signal and can give rise to kinetic facets of the ligand-analyte interaction. Schlecht et al. compared an RNA aptamer and an antibody for thrombin sensing by using a nanometer gap-sized electric resistance biosensor. They found that both ligands showed equal suitability for the highly specific sensing of their analyte.
Their device has a multiplexer approach, enabling the parallel read-out of five detector elements. This opens up the possibility to use these detectors for the elimination of background signals and the simultaneous sensing of different analytes by immobilizing their respective ligands on separate electrodes.
For electric resistance methods, normally, a negative read-out signal can be found in effect of an increase in electron transport resistance. However, Rodriguez et al., 2005 described the set-up of an impedance-based method exhibiting a positive read-out signal by making use of the alteration of surface charge from negative to positive upon the target protein binding (at proper pH).
A very similar approach, also depending on electrostatic interactions, was made by Cheng et al., 2007. A DNA aptamer for muramidase was immobilized on gold surfaces by means of self-assembly and [Ru(NH3)6]3+ attached to the DNA phosphate anchor via electrostatic interaction. The surface density of aptamers can be determined by measuring the [Ru(NH3)6]3+ reduction peak height in the cyclic voltammogram.
Upon target binding of muramidase to the aptamers, the surface-bound [Ru(NH3)6]3+ cations are released. This can be detected as a reduction in the integrated charge of the reduction peak. The hindrance of the redox reaction of K3Fe(CN)6 on a gold surface due to an increased density of the covering layer by binding of the immobilized DNA aptamer with its target thrombin was used as a signal for the binding reaction. The signal was measured by cyclic voltammetry. The aptasensor for thrombin is reusable and allows measurements in the relevant analytical scope for clinical applications.
Another label-free method is to use intercalators that bind to duplicate isolated parts of the aptamer. If these parts are close enough to the electrode, the intercalators can function as reporters. Upon binding and the subsequent conformational changes, the intercalator can be released, producing a negative response.
For example, an aptamer for thrombin was immobilized on a gold electrode. Methylene blue (MB) intercalates into a double-strand part and will be released upon target binding due to the conformational change of the aptamer. The MB cathodic peak current in the differential pulse voltammogram decreases with increasing thrombin concentration.
These techniques described above are label-free, that is, neither the bioreceptor nor the target has to be covalently labeled with index molecules, and this, therefore, omits a further step in the production process of the detector. In contrast, many electrochemical aptasensors rely on the labeling of the bioreceptor with a reporter unit. For example, aptamers can be labeled at both terminals. At one terminal, a moiety for immobilization at the surface is tethered to the aptamer, and at the other terminal, the reporter.
The electrode surface is then covered with a layer of these aptamers. Upon target binding, the mobility of the aptamer and/or the density of the layer are altered due to beacon-like conformational changes. This results in a smaller or greater distance of the reporter unit from the electrode, leading to increased or decreased electron transport, respectively.
Sandwich assays rely on the possibility that more than one aptamer can be generated for one protein marker. One aptamer, attached to the detector surface, binds the marker at one antigenic determinant. The second aptamer, directed to a different antigenic determinant, is labeled with the newsman, e.g. (PQQ) glucose dehydrogenase.
Binding of the second aptamers to the marker brings the newsman in propinquity to the detector surface. After a washing measure, the binding is detected (in this instance, by amperometry after addition of glucose as a substrate for (PQQ) glucose dehydrogenase), leading to a positive read-out signal via the redox mediator 1-methoxyphenazine methosulfate.
Optical aptasensors
Optical transduction methods in aptasensors comprise, for example, the use of surface plasmon resonance, evanescent wave spectrometry, as well as fluorescence anisotropy and luminescence sensing. Surface plasmon resonance (SPR) and evanescent wave-based biosensors rely on the alteration of optical parameters upon alterations in the layer closest to the sensitive surface. Since the binding of, for example, proteins to a receptor layer of those biosensors changes the refractive index of the layer, the event of binding can be detected and quantified in a label-free manner.
Examples for the usage of surface plasmon resonance biosensor sensing of the several markers adhering to the bioreceptor – the aptamer (in most instances thiolated for the immobilization at gold surfaces by self-assembly). Thrombin was captured by a DNA aptamer immobilized at Biacore® chips. Several parameters like incubation time, incubation temperature, effect of immobilization orientation, etc. were extensively studied and optimized.
IgE was captured by a DNA aptamer with a sensing bound of 2 nanometers and a linear range of sensing from 8.4 to 84 nanometers using a combination of the methods of SPR and fixed-angle imaging. HIV-1 Tat protein was captured by an RNA aptamer with a linear sensing range from 0 to 2.5 ppm using a Biacore X™ instrument. Due to the inherent sensitivity of RNA to nucleases, all equipment was freed from RNases prior to preparation of the detector chips and measurements.
Mass-sensitive aptasensors
Microgravimetric methods on piezoelectric silica crystals are based on the alteration of the oscillation frequency of the crystal upon mass alteration at its surface due to receptor-target binding (quartz crystal microbalance, QCM). This alteration of oscillation frequency is the signal that is detected. With this method, a label-free sensing of the marker is possible. However, the use of “weight labels” – e.g. aptamer functionalized Au nanoparticles – for the elaboration of the binding reaction on the QCM surface seems useful.
Quartz crystals were coated with gold layers and streptavidin was later immobilized. Biotinylated aptamers were then added and used as the receptor layer. DNA aptamers were used for the sensing of IgE with a sensing bound of 100 µg/L and a linear sensing range from 0 to 10 mg/L. HIV-1 Tat protein was detected using RNA aptamers as receptors. Detection bounds of 0.25 ppm and 0.65 ppm with linear sensing ranges of 0-1.25 ppm and 0-2.5 ppm, respectively, were achieved.
Potentiometric aptasensors
Potentiometric detectors are based on the measurement of a difference in potential between the working and reference electrode caused by a difference in analyte concentration. Field-effect transistors belong to the category of potentiometric detectors. Carbon nanotube field-effect transistors (CNT-FETs) are among the most promising candidates to potentially replace CMOS (complementary metal-oxide-semiconductor) engineering by further miniaturization.
The semiconducting behavior of CNTs is the chief reason for the endeavor to construct CNT-FETs. Aptamer-modified CNT-FETs for the sensing of IgE were constructed and compared to CNT FET biosensors based on a monoclonal antibody (mAb) against IgE. 5′-amino-modified 45-mer aptamers and IgE-mAb were immobilized on the CNT channels, respectively. The amount of net source-drain current increased in dependence on the IgE concentration after IgE debut on the aptamer-modified CNT-FETs.
The sensing limit of 250 attomolar and additive dynamic range of 250 attomolar to 20 nanometer was determined. The IgE-mAb detector showed only a small change in the net source-drain current at 0.2 and 1.8 nM IgE. The aptamer-modified CNT-FETs displayed a better performance for IgE sensing under similar conditions than the monoclonal antibody-based CNT-FET.
Aptamer biosensor for protein sensing:
- Target Protein
- Aptamer
- Type of Sensor, Reporter
- Unit of measurement
- Thrombin
- Deoxyribonucleic acid beacon
- European Union, differential pulsation
- voltammetry, methylene
- bluish intercalator
- Thrombin
- Deoxyribonucleic acid
- European Union, electric resistance
- spectrometry, [ Fe ( CN ) 6 ] 3-/4-
- Thrombin
- Deoxyribonucleic acid thiolated/
- biotinylated
- European Union, differential pulsation
- polarography,
- p-nitroaniline/peroxidase/HRP
- Thrombin
- Deoxyribonucleic acid thiolated/
- biotinylated
- optical, SPR
- Lysozyme
- Deoxyribonucleic acid
- European Union electric resistance
- spectrometry, [ Fe ( CN ) 6 ] 3-/4-
- Immunoglobulin e
- Deoxyribonucleic acid thiolated
- optical, SPR
- Immunoglobulin e
- Deoxyribonucleic acid
- European Union electric resistance
- spectrometry, array
Drumhead
The use of aptamers as new biological receptors can accelerate the development of biosensors of practical relevance. Because of their exceptionally high stability, selectivity, and sensitivity, aptasensors have the potential to overcome the deficient functional and storage stability of most biosensors (also some exceptions like glucose and lactate biosensors that are very well established in the market).
This review shows that a large variety of biosensor principles (e.g., electrochemical, optical, mass media) is available for the use of aptamers as biological receptors. However, only for a few proteins (thrombin, muramidase, IgE, and some others) have aptasensors been described. The more aptamers for proteins that are developed and characterized, the more aptasensors will be developed in the future.
