The rapid technological progress of the past couple of decades in the fields of biosensors and microfluidics [1
], has allowed the originally envisioned democratization of healthcare of the early 1990s [2
] to be closer to realization than ever. A broad range of different biosensing concepts and microfluidic structures currently allows the qualitative or quantitative detection of various biomarkers of clinical interest. With DNA biomarkers [3
] increasingly being identified and associated with a very broad spectrum of clinical diseases (e.g., cancer, infectious disease, antimicrobial resistance, etc.), intense scientific effort has been focused on their sensitive and specific detection. With the ultimate goal being affordable, easy to use and small footprint DNA diagnostic microsystems, microfluidic structure integration for sample pre-processing and automated reagent handling are some of the critical aspects being investigated currently [5
]. Simplicity in device functionality is critical, aspiring for robust and reliable diagnostic microsystems; to this end, simplification of the biosensing assay by removing the need for any additional labelling step [6
] can provide a step-change in the development of tests ready for mass deployment.
Therefore, in this review our goal is to present recent developments in label-free DNA biosensors, with a particular focus on integration with microfluidic structures. We classified the microfluidic devices based on the integrated microfluidic structure complexity, starting from the simplest approach of flow-cell integration. Given the number of interesting flow-cell integrated structures, they were sub-classified using the biosensing principle employed. We then proceeded to more complicated microfluidic structures for label-free DNA biosensing platforms, higher integration examples, and state-of-the-art nanofluidic-based DNA sequencers. We conclude with the commercialization efforts currently being pursued in the field. It is worth noting that our definition of label-free detection involves techniques that do not require the attachment of any additional molecule on the target DNA sequence; hence techniques involving the use of reagents diluted in solutions for measurement purposes, are still considered to be label-free.
4. Higher Integration Platforms
With the realization of all the previously reported efforts, the true potential of Lab-on-Chip technology can be realized in sample-in-answer-out devices. The whole analytical procedures described earlier (including sample pretreatment, sample manipulation, separation, reaction, and detection), can be integrated into a single chip platform. Over the past years, several partially integrated systems have been presented in literature, with increasing complexity and degree of integration in more recent studies. An in situ electrochemical (EC) detection method in a microfluidic flow-through EC-quantitative PCR (FTEC-qPCR) device was developed, where both the target DNA amplification and subsequent EC detection of the PCR amplicon were performed simultaneously in the same device [55
]. An electroactive reporter (methylene blue) was used for in situ EC detection of the PCR amplicon. Woolley et al. demonstrated an integrated microfluidic device, which permitted the direct integration of microfabricated PCR and capillary electrophoresis (CE) components on a single micro-device [56
]. The rapid thermal cycling capabilities of microfabricated PCR devices with high-speed DNA separations provided by microfabricated CE chips, was the critical microfluidic addition. A microfabricated device for cell isolation was presented by Wilding and co-workers [57
], with the benefit of integrating whole blood sample preparation on-chip for subsequent nucleic acid amplification. White blood cells were isolated from whole blood in silicon–glass microchips using filters, formed by an etched silicon dam spanning the flow chamber. The direct DNA amplification was done using polymerase chain reaction from the white cells isolated on the filters. Papadakis et al. 2018 [58
] reported a micro-nano-bio acoustic system for the detection of foodborne pathogens in real samples with a highly integrated microfluidic system. The authors used immuno-magnetic beads to capture cells, and subsequently demonstrate efficient DNA amplification using the Loop Mediated Isothermal Amplification method (LAMP) and acoustic detection in an integrated platform. However, there was still a need to further integrate more of the required detection steps in a single microfluidic chip. Liu et al. presented a highly integrated plastic monolithic device, where PCR amplification, DNA hybridization, and a post-hybridization wash have been integrated in a single, low-cost, disposable monolithic device, containing all of the necessary fluidic channels and reservoirs [59
]. Very recently, Nguyen et al. developed a unique highly integrated microfluidic device, performing not only the amplification of DNA of Salmonella
spp. within 30 min, but also the immediate subsequent measurement of DNA amplicon by the SPR fiber sensor part [60
]. The novelty was the integration of a polymerase chain reaction (PCR) microdevice and a surface plasmon resonance (SPR) optical fiber sensor into an inline all-in-one device (Figure 10
). The authors proposed that the detection instrument could be further miniaturized by incorporating a miniaturized light source/photodiode.
Another multiplexed integrated label-free DNA detection platform was proposed by including two functional modules, i.e., a multiplexed PCR module for amplification of nucleic acid targets, and a multiplexed silicon nanowire (SiNW) module for sequence determination [61
]. The PCR module consisted of a microfluidic PCR chamber and an electrical controller. A control SiNW was implemented to eliminate background interference. The detection module demonstrated a 10-fold change in the magnitude of differential current when the target DNA was injected. The low sample consumption, high sensitivity, and high specificity, rendered it a potential point-of-care (POC) platform to assist doctors in reaching a yes/no decision for infectious diseases.
To eliminate the time-consuming PCR step, Medina-Sánchez et al. reported an ultrasensitive label-free DNA biosensor with fully on-chip integrated rolled-up nanomembrane electrodes, for avian influenza virus subtype H1N1 DNA detection, with an attomolar detection range for miniaturized sensors without amplification. The electrodes were prepared via sequential deposition of strained nanomembranes onto a sacrificial layer, which was then selectively dissolved, resulting in the self-roll-up of the microtubular electrodes [62
Haber et al. [63
] recently integrated on-chip DNA hybridization and real-time PCR using an LSPR-based sensor. They outlined the development of a novel microfluidic sensor for the implementation of qPCR, employing a piezo-electric pumping microsystem. Complementary DNA probes were immobilized onto the surface of nanoprisms on the surface of glass attached via poly-l
-lysine linkers. DNA hybridization was then measured using label-free LSPR imaging for real-time results (Figure 11
). A limit of detection of 5 fg/µL was demonstrated for E. coli
DNA (approximately 300 bacteria per mL). Detection of target DNA was achieved within 15 min of PCR initiation, which promises the type of rapid diagnosis required for POC devices.
Droplet-based microfluidics are also an ideal integration platform for applications requiring high-throughput analysis [64
]. These systems offer the unique advantages of automated compartmentalization of reagents in multiple picoliter volume drops, along with the possibility to perform in a programmable way, multiple combinations of reagents rapidly. Many such platforms have been integrated with label-free biosensors. Indicatively, Malic et al. [65
] incorporated Surface Plasmon Resonance Imaging (SPRi) DNA sensors, achieving 500 pM LOD. Hsieh et al. [66
] employed a molecular beacon fluorescence detection approach achieving 500 fM LOD, whilst Ebrahimi et al. [67
] integrated non-Faradaic electrochemical impedance spectroscopy biosensors in a droplet microfluidic platform, reaching aM-scale DNA detection.
5. Nanopore Technology for Label-Free DNA Sequencing
Nanopore-based systems are nanofluidic devices for label-free sequencing of DNA molecules, drawing inspiration from the biological nanopores present in cellular membranes [68
]. The physical concept can be accurately described as a micro-Coulter counter for DNA bases; where a voltage is applied between the nanopore inlet and outlet, and the polynucleotide bases passing through the pore modulate the ionic current proportionally, allowing the sequential identification of each base in the analyzed strand.
The first nanopore sensor mimicked this natural presence of nanopores in biological membranes (Figure 12
), embedding a single α-hemolysin protein in a lipid bilayer to exploit its inner diameter compatibility with DNA molecules. Solid state nanopores (e.g., SiNx
) soon substituted the biological original protein nanopores, owing to the instability of the supporting lipid bilayer under chemical, electrical, or physical variations. The introduction of solid state nanopores also allowed more versatility in their fabrication, enabling variable but controlled diameters and geometries, even in sub-nanometer resolutions.
This advancement towards solid state nanopores has given rise to a variety of nanofabrication efforts [69
] exploiting different material properties, aiming to achieve the optimum nanopore resolution, thickness, and reduction of DNA passage speed through the nanopore. The combination of e-beam lithography with reactive ion etching, focused-ion beam, and helium ion beam methods, have been presented in literature with impressive results. Currently, the prevailing technique lies with transmission electron microscopy (TEM) focused ion beam, allowing real time control of nanopore formation in the order of 2 nm diameters. Another interesting alternative fabrication technique involves glass capillary nanopores (also known as glass nanopipettes) [70
]. This approach proves particularly advantageous in terms of stiffness, manufacturability, and cost-effectiveness, with shrinking of the device, making it ideal for DNA translocation studies [72
The nanofabrication of a complete, high performing nanopore-based DNA sequencing system is currently the main focus of researchers [73
], with the objective of combining solid state nanopores, sensing nanoelectrodes for the detection of the electrical signal and sample delivery nanofluidics into a rapid and reliable DNA sequencing microchip. The commercial success of Oxford Nanopore Ltd. (see the following paragraph), launching MinION as the first portable commercial label-free sequencer [75
], has led researchers to assess its performance in several real-life applications, and to try to identify its current limitations [76
]. The initial results look very promising in terms of throughput and analysis time, nonetheless, further technological development is required to reduce the error rate to match benchtop equipment performance.
6. Commercial Systems
In the commercial arena, one of the most widely used techniques for label-free biosensing of DNA is IonTorrent, now part of Thermo Fisher Scientific, which uses semiconductor technology for DNA sequencing. The process involves the library preparation by DNA fragmentation and attachment to a magnetic bead, where a single fragment is amplified. Beads carrying copies of label-free ssDNA are loaded onto the sequencing chip, where each of the beads is captured in an individual well containing a miniaturized ion-sensitive field-effect transistor (ISFET) pH sensor. The chip is flooded with a solution containing a single nucleotide type and where a match is present, the nucleotide is incorporated, releasing a hydrogen ion which lowers the pH of the solution in the well, and is detected by the built-in ISFET. If more than one base is present in succession, the signal gets proportionally higher. Different nucleotide solutions are sequentially introduced onto the chip, enabling the sequence determination. Millions of wells are simultaneously recording nucleotide incorporation, enabling parallel reading and massive scalability [79
]. IonTorrent technology is currently covering multiple applications from targeted DNA sequencing, microRNA sequencing, to bacterial and viral typing [81
Oxford Nanopore Technologies, a spinout from the University of Oxford, brought sequencing technology to its users as a USB powered pocket-sized device called MinION. To start off, a DNA library can be prepared in 10 min, and data can be observed in real time during sequencing. After commercialization of MinION in 2014, the GridION and PromethION systems have been developed, enabling up to 5 and 48 parallel MinION flow cells, respectively [82
]. The MinION device has been used to sequence human genome [83
], and has been especially useful in monitoring epidemic diseases at remote locations, due to is portability and sequencing speed [84
]. The company is currently working on an even smaller portable device, which will be able to perform sequencing whilst connected to a smartphone [82
In the effort to bring nucleic acid detection to remote locations, QuantuMDx is developing the Q-POCTM
, a handheld device compatible with disease specific cartridges [85
], capable of sample preparation, DNA extraction, amplification, and detection in 10–20 min, from multiple biological samples [86
]. They envision a wide area of applications, ranging from drug resistance analyses for tuberculosis, infectious disease identification, such as STIs and HPV, to metabolism genotyping to identify the optimal warfarin dosage [87
]. The company is developing two detection approaches [88
]: An optical based strategy using fluorescent markers and an electrochemical one, based on label-free nanowire field effect transistors [89
]. One of their goals is to create a world-wide map to monitor pathogen evolution, in the effort to prevent global outbreaks [86
]. To achieve this, they are aiming to use nanowire FETs to sequence pathogens DNA in a portable device [89
]. However, to the best of our knowledge, the Q-POCTM
device, which is planned to be launched in 2018, uses fluorescence detection [86
Another company trying to reach the diagnostic market is Atlas Genetics, a spinout from the University of Bath, which offers their CE-marked io®
system for detection of STIs and other infections. The io®
instrument is a benchtop device, in which a single-use cartridge is inserted, and the result is obtained within 30 min. First, the sample is introduced into the cartridge, where DNA is extracted and amplified via PCR [90
]. The detection chamber includes target complementary probes, linked to a ferrocene tag and a double strand specific nuclease [91
]. When a label-free target DNA hybridizes with a detection probe, nuclease cleaves off the ferrocene tag, which is detected electrochemically, using differential pulse voltammetry [92
]. Including multiple detection chambers in a single cartridge, and engineering ferrocene labels and their electrochemical properties [93
], grants the io®
system a high capacity for multiplexing. They have published multiple studies using clinical material for diagnosis of pathogenic Candida
], Chlamydia trachomatis
], and Trichomonas vaginalis
Recently DNAe, a spin-out from Imperial College London, presented a semiconductor sequencing based benchtop device called LiDiaTM
, which can accurately detect pathogen DNA and identify from bloodstream infections to antimicrobial resistance. During the process, the blood sample is introduced into a disposable cartridge and loaded to the reader. Target extraction, concentration, and isolation, along with genomic analysis on a chip is performed in a single cartridge [98
]. The amplification reaction occurs on the chip integrated with embedded heaters and ISFETs. As specific primers recognize the label-free target DNA, the amplification reaction initiates. With each nucleotide being incorporated to an amplicon, a proton is released, lowering the pH in the solution. Therefore, amplification reaction is detected in real time by the ISFET [99
]. DNAe are currently testing the device with clinical samples with a plan for CE application in 2018. One of their main applications is detection of sepsis, where their device can provide information about the appropriate antimicrobial treatment within 3 h [98
7. Conclusions and Future Outlook
In this article, we reviewed recent technological progress in the field of microfluidic-assisted label-free DNA biosensing devices. We mainly focused on label-free highly integrated biosensor devices, which are suitable for point-of-site detection, such as medical diagnostics, biological research, environmental monitoring, and food analysis. Optical, electrochemical, mass-based label-free biosensing approaches are currently being pursued for microfluidic integration, each featuring particular advantages and disadvantages. Mass-based methods can provide very efficient detection for heavier molecules, nonetheless, they can prove less competitive for smaller molecules, such as short chain oligonucleotides. On the other hand, optical approaches can provide very low-limits of detection even for very small molecules; however, they suffer from their elaborate instrumentation requirement in terms of platform miniaturization. Electrochemical biosensors offer the advantage of combined low limits of detection and minimal, low-power instrumentation requirements. All sample preparation microfluidic modules enabling a sample-in-answer-out system (i.e., cell-isolation, DNA extraction, DNA amplification), have been individually demonstrated in the literature; nonetheless, very few efforts for multiple module integration have been reported so far. Multiplexed DNA amplification modules, droplet-based microfluidics, and nanopore-assisted sequencing, clearly indicate the technical feasibility of high-throughput and high specification portable systems in the future. Currently via nanopore technology, it is possible to have DNA sequencing at the point-of-need, at a throughput of 10–20 G bases per 48 h. Whilst such a sequencing throughput is impressive, there are numerous diagnostic applications where full genome sequencing is not required. In such cases, microfluidic platforms offering sample-in-answer-out operations, with time to result of a few minutes, may prove more practical in real-life practice, both for Point-of-Need use in Centralized healthcare systems and for low-resource settings.
Building on more mature technologies in microfluidic DNA systems, several commercial efforts are already promising to launch in the immediate future sample-in-answer-out systems, with specific medical diagnostic applications. A major challenge to be overcome pertains to the cost-effective integration of highly sensitive biosensors in portable devices, or even in equipment-free systems. At the moment, a seamless mass-manufacturing approach for integration of all the required components is not commercially available, with each diagnostic company pursuing their own strategy. Moreover, the exploitation of sensing approaches requiring elaborate or expensive instrumentation can present obstacles in portability, along with the large power requirements associated with heating on modules incorporating DNA amplification. With most practical applications demanding the quantification of multiple DNA strands simultaneously, both assays and devices allowing multiplexed detection need to be developed. Finally, device development should be carried out based on real physiological samples early on, assuring device compliance with international regulatory frameworks and biomedical device standards.