Developments in nanobiosensors: recent research

Developments in nanobiosensors: recent research

Article Type: Nanosensor update From: Sensor Review, Volume 32, Issue 4

Biosensors find uses in fields such as healthcare and medical research, environmental monitoring, law enforcement and homeland security and rely on a reaction between the target compound and a biological “agent” being detected by an electrical, optical, mechanical or other transduction technique. As with all other classes of sensor, biosensors are being developed which incorporate nanomaterials and nanosensing concepts, frequently with the aim of enhancing performance. All of the well known nanomaterials and structures have been incorporated into prototype nanobiosensors, e.g. CNTs, metallic nanoparticles, graphene, quantum dots (QDs) and nanowires. Nanobiosensors are the topic of a global research effort and some industry commentators argue that of all the classes of nanosensors under development, nanobiosensors offer amongst the greatest long-term commercial prospects. In part this reflects the fact that many biosensor applications require high sensitivities and while many biosensing principles are inherently very sensitive, this can frequently be enhanced further through the use of nanomaterials, often as a consequence of their exceptional surface area-to-volume ratios which boosts the number of available reaction sites. Some examples of recently reported nanobiosensor developments are considered below.

As with the previously discussed NEMS work, research at Cornell University, reported in 2012, involves a resonant transduction concept. An optothermally (laser) excited NEMS resonator has been fabricated which comprises ordered, vertical silicon nanowire arrays above a Si/SiO₂ bilayer membrane. Single-stranded “probe” DNA molecules are bound to the nanowires which react with and bind to “target” DNA, causing a mass increase which alters the membrane’s resonance frequency. The sensor exhibited a DNA sensitivity of 500 aM (10⁻¹⁸ M) which was achieved due to the resonator’s high surface area-to-volume ratio (10⁸ m⁻¹) and mass-per-area resolution (1.8×10⁻¹² kg m⁻²). Further, the nanowire array acts as a photonic crystal which shows strong light trapping and absorption over a broad range of wavelengths and allows over 90 per cent of the incident laser light to be absorbed.

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Figure 6 Schematic of a single TNT molecule on the surface of a CNT

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Figure 7 Analytical curve obtained with the CNT-AChE biosensor for different concentrations of chlorphenvinphos in a phosphate buffer

CNT-based nanobiosensors have been studied widely and frequently rely on functionalisation of the tube with a bioactive component and detecting the electrical or optical change resulting from the reaction with the target compound. As an example, unique work at MIT has used bombolitin II, a variant of a bumblebee venom-derived amphiphilic peptide, as the bioactive compound to detect trace levels of nitro-aromatic compounds such as the explosives RDX and TNT. The bombolitins are bound to the CNTs which are naturally fluorescent at NIR wavelengths. When target molecules react with the bombolitins they cause a shift in the wavelength of the fluorescence. Further, compounds such as TNT decompose in the environment creating other compounds and these derivatives could also be identified with this type of sensor. In addition, the group has found that an oligonucleotide of single-stranded DNA with the sequence ss(AT)₁₅ imparts optical selectivity for TNT via intensity (rather than wavelength) modulation. This technique is exceptionally sensitive and can detect a single TNT molecule (Figure 6). As with conventional biosensors, many nanobiosensors have been developed for the determination of water-borne pesticides and a recent example is work by the Brazilian Federal Universities of Uberlandia and Sao Carlos. Here, the enzyme acetylcholinesterase (AChE) was combined with CNTs to form a paste which was used to produce an amperometric electrode. The reaction between AChE and organophosphate (OP) compounds is very well characterised and in this work the target was the OP pesticide chlorphenvinphos. An electrochemical cell was constructed using a conventional three-electrode system: an Ag/AgCl in KCl reference electrode, a platinum counter electrode and the CNT-AChE biosensor which acted as the working electrode. Amperometric measurements over the chlorphenvinphos concentration range 4.90×10⁻⁷ to 4.76×10⁻⁶ M showed a response as shown in Figure 7 and the limit of detection was 1.15×10⁻⁷ M. These results agreed well with data derived from tests conducted by a standard spectrophotometric method, showing an error of <3 per cent. Most importantly, because CNTs promote electron transfer reactions due to their electronic structure, high electrical conductivity and redox active sites, the sensor was produced without the introduction of redox mediators. Technologically similar research, also aimed at detecting a water pollutant, nitrates, was reported by workers from the Turkish Gebze Institute of Technology in 2011. This was based on an amperometric electrode fabricated from a thin film composed of polypyrrole, CNTs and the enzyme nitrate reductase. This sensor showed a linear response over the nitrate concentration range 0.44-1.45 mM, a sensitivity of 300 nA/mM and a limit of detection of 0.17 mM. Many other CNT/enzyme- and nanoparticle/enzyme-based nanobiosensors have been reported for both water pollutants and clinical analytes and are representative of the growing body of work which aims to improve the performance of electrochemical biosensors through the use of nanomaterials.

Nanobiosensors based on QDs modified with a range of different biologically active compounds have been reported widely in the recent literature. Through the use of these compounds and various bioconjugation strategies, it is possible to use QDs as the sensing elements for all manner of biorecognition events. The modulation of QD luminescence allows the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer, charge transfer quenching and electrochemiluminescence. In the medical/biological context, there are many potential applications such as the detection of small molecules using enzyme-linked methods, or using aptamers (oligonucleic acid or peptide molecules that bind to a specific target molecule) as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridisation assays; and assays for protease or nuclease activity. As an example of a recent medical development, in 2011 workers from the National Tsing Hua University in Taiwan reported the use of CdSe/ZnS QDs to detect human serum albumin (HSA), a critical protein produced by the liver and used to test liver function. The QDs were modified with monoclonal anti-HSA and a simple optical system, comprising a diode laser operating at 405 nm, an optical lens, a 515 nm long-pass filter and a silicon photodiode, was used to detect fluorescence and convert it into a photocurrent. The current intensity could be used to quantify the HSA concentration and the limit of detection was found to be approximately 3.2×10⁻⁵ mg mL⁻¹. In 2011, workers from the University of Toronto Mississauga reported the use of CdSe/ZnS QD-FRET technology to develop solid-phase nucleic acid hybridisation assays. The instrumentation used is shown in Figure 8 and it is anticipated that the incorporation of QD-FRET assays in microanalytical systems could yield detection limits that are orders of magnitude better than those observed with macroscopic systems. QDs have also been used in sensors for the detection of pesticides. In 2011 workers from China’s University of Jinan and Nanyang Technological University, Singapore, used a combination of CdTe QDs and molecularly imprinted polymers to detect deltamethrin. Measuring the chemiluminescent intensity from the QDs allowed this pesticide to be determined over the linear range 0.053-46.5 μg mL⁻¹, with a detection limit of 0.018 μg mL⁻¹.

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Figure 8 Instrumentation arrangements

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Figure 9 Approaches to preparing graphene-based electrochemical biosensing platforms

As reported in previous Nanosensor updates, the sensing capabilities of graphene are the topic of global investigations and since its discovery in 2004, many groups have studied its potential in biosensing. Prototype graphene-based nanobiosensors based on electrochemical, optical and other principles have been developed, sometimes in combination with other nanostructures such as QDs and magnetic nanoparticles (Figure 9). They have been used to detect many clinical and other analytes, including glucose, hydrogen peroxide, nicotinamide adenine dinucleotide (NADH), dopamine, DNA, cancer biomarkers and ethanol. Details of these developments will be covered in a future update.