A review of microfabricated electrochemical biosensors for DNA detection
Introduction
Biosensor is a commonly used and broad term, which describes almost any sensor with a biological component. These tend to take the form of a layer of complex molecules attached to a sensor, where target binding or a change in the molecules which comprise the sensing layer causes a physical change which can be measured by the underlying device. This ensures bio-recognition or signal specificity and enables the physical sensor to detect the presence of biological targets that would typically be unavailable to it, such as specific proteins, DNA/RNA sequences, and bacteria species. Numerous different biological detection methods have arisen, with popular ones including: surface plasmon response (SPR), Raman and surface-enhanced Raman spectroscopy (SERS), vibration of mechanical cantilevers, and electrochemical measurements (Arlett et al., 2011; Hansen and Thundat, 2005; Homola, 2003; Ngo et al., 2015; Šípová and Homola, 2013; Wang, 1999; Wolfbeis, 2008). Electrochemical methods have received widespread attention due to the relative simplicity and cost-effectiveness of the required set up in addition to their ease of miniaturisation (Drummond et al., 2003; Ferapontova, 2017; Grieshaber et al., 2008).
DNA biosensors allow for the detection and quantification of specific DNA sequences. Despite already proving a very useful analytical tool, it is the clinical setting where these devices have the potential for the highest impact. The ability to rapidly determine the presence of a certain DNA sequence in a clinical sample means rapid diagnoses of almost any disease from non-communicable (e.g. cancer) to infectious (e.g. HIV, malaria and sepsis), as well as the presence of poisons like pesticides and is an important means for determining the presence of antibiotic resistance (Liu et al., 2019; Kumar et al., 2019; Diculescu et al., 2005; Bartosik and Jirakova, 2019). Combined with the benefits of microfabrication and lab-on-a-chip approaches, it is not surprising that electrochemical DNA sensors for biomedical applications are a popular area of research.
The operation of an electrochemical DNA sensor generally starts by forming a self-assembled monolayer (SAM) of single stranded (ss)DNA on the surface of an electrode. This DNA has been designed as a genetic recognition sequence which will only bind to a specific target strand of interest. The DNA modified electrode is incubated in a sample solution and any target DNA in the solution is hybridized with the probe strand to give double stranded (ds)DNA on the electrode surface. In a complex mixture, such as a clinical sample where background DNA is present, any of the complementary target sequences which are present will bind to the probe DNA immobilized on the electrode. Measurements of an electrochemical parameter which is dependent on the state of the monolayer are performed before and after this hybridization step. These can include the changes in double layer capacitance, charge transfer through the DNA film via a solution based redox mediator, or electron transfer currents from a redox label bound to the probe sequence or target DNA. An example of a commonly used detection method is presented in Fig. 1 (Li et al., 2017a, Li et al., 2017b; Grieshaber et al., 2008; Wang, 2006). A negatively charged redox molecule is measured using electrochemical impedance spectroscopy (EIS) at an electrode surface, shown in Fig. 1 (a). A ssDNA probe layer is assembled on the electrode surface and increases the charge transfer resistance of the reaction, as in Fig. 1 (b). After hybridization with the target in Fig. 1 (c), the amount of DNA in the film increases, further raising the charge transfer resistance, shown in Fig. 1 (d).
Many studies with electrochemical DNA sensors focus on addressing challenges such as improving sensor specificity and limits of detection. However, even once these goals are satisfactorily achieved the further problem of manufacturing the sensor becomes apparent. This is especially pertinent as many biosensing strategies have involved modification of electrodes with materials such as nanoparticles and graphene, or even bespoke polymers. The difficulty with these devices is their capacity for production, not only on a large scale but also with repeatable performance. A solution to this is the use of microfabricated sensors (Wang, 2000). At present, microfabrication processes are quite mature and responsible for the mass manufacture of millions of complex electronic components every year. The benefits of developing a sensor system with such processes in mind not only enables the large-scale production of nominally identical sensors, but the integration of them with a myriad of different technologies (Blair et al., 2015; Marland et al., 2018.). This includes wireless and smartphone technology, which is covered in a recent review on wireless chemical and biosensors by Kassal et al. (2018). This helps realize the goal of lab-on-a-chip style systems, where the sensor is integrated with signal processing and read out electronics on a single platform. Then the chip can be combined with microfluidic packaging for sample processing and delivery (Buchoux et al., 2017; Lafleur et al., 2016). The end result is a complete point-of-care system which can be cheaply manufactured and easily used without specialized training. This review will focus on the current state of biosensors which are compatible with microfabrication processes. The goal of this review is to cover the recent literature in this area and provide readers new to the field with a roadmap for fabrication of their own systems, which best suit the requirements of their application. Other forms of microfabricated DNA sensors exist such as Field-Effect Transistor (FET) based sensors and those based on selective nanopores. These will not be investigated here and an interested reader is pointed to several relevant reviews (Howorka et al., 2001; Mattiasson and Hedström, 2016; Veigas et al., 2015). Table 1 contains a list of other recent reviews that expand areas touched on in this work giving the reader an oversight into the field of biosensors, as well as showing where this review fits into the literature. This review will first look at work involving common thin film materials used to make DNA biosensors, including their benefits and drawbacks. The effects of miniaturisation will then be investigated, looking at research conducted using microfabricated microelectrodes and nanoelectrodes as well as previous attempts to integrate these onto complimentary metal–oxide–semiconductor (CMOS) chips. Microfluidics and chip packaging are then looked at, followed by investigating the uses of different electrochemical measurement techniques. Finally, a summary of the direction of microfabricated DNA biosensors will be presented.
Section snippets
Gold
The most common material typically used for microfabricated biosensor electrodes is gold. Such thin films are usually sputtered or evaporated between thicknesses of 10–500 nm (Díaz-Serrano et al., 2011; Hong et al., 2018; Hsu et al., 2016; Soraya et al., 2018). Full microfabrication processes on silicon wafers can be very expensive and many groups instead use glass slides, for example Capaldo et al. used a lift-off process to pattern gold on a microscope slide and then used SU-8 photoresist as
Microelectrodes
It has long been known that microelectrodes offer many analytical advantages over macro-scale electrodes. The hemispherical diffusion profile typical of microelectrodes in combination with the small surface area of the sensor yields a higher signal to noise ratio, higher current density, and simpler analytical treatment for Faradaic processes (Bard et al., 1980; Corrigan et al., 2014; Forster, 1994; Stulík et al., 2000). Despite this, thin film microelectrodes are not as frequently used as DNA
EIS
When implementing a microfabricated electrochemical biosensor system it is possible to utilize the same electroanalytical techniques employed for macro scale devices and electrodes produced using other approaches, e.g. screen printing. For microelectrode and nanoelectrodes the expectation is that enhanced sensitivity will be achieved through the favorable electro-analytical properties which arise from employing electrode sensors with such small dimensions. EIS is a versatile and sensitive
Summary and conclusions
Table 2 presents a selection of thin film biosensors from literature, showing aspects of their design, performance, and characterization. These examples demonstrate that thin film DNA biosensors have attained a whole range of sensitivities and performances in complex and simple media. It is important to remember that comparisons between these sensors must be done carefully as they fulfill a myriad of different functions with differing requirements (indeed a recent piece by the editorial board
Future perspectives
One particularly obvious difficulty for future mass production of electrochemical DNA biosensors highlighted by Barbaro et al., is that most utilize metals such as gold, platinum, or silver (Barbaro et al., 2012). This can be a problem for scaling up their manufacture as these materials are incompatible with CMOS foundry fabrication. This is demonstrated by the many groups who have CMOS chips fabricated in an external foundry, and then must apply materials such as these in-house using
Acknowledgements
This work was supported by a British Council Institutional Links grant, ID 20180209, under the Newton-Katip Çelebi Fund partnership. The grant is funded by the UK Department of Business, Energy and Industrial Strategy (BEIS) and Tubitak and delivered by the British Council. For further information, please visit www.newtonfund.ac.uk.
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