Advances in Medical Diagnostics Using LoC and LoPCB Technologies
Introduction
“The Coronavirus: A Global Pandemic” has become the universal headline. As of mid-March, the World Health Organization characterized the coronavirus as a pandemic, which had already spread to almost 150 countries, areas, and territories, with hundreds of thousands of confirmed cases.
Coronaviruses (CoVs) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as the Middle East respiratory syndrome (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV). COVID-19 is a new strain that was discovered in 2019 and had not been previously identified in humans.
In an outbreak of a new virus, it is imperative that epidemiologic and clinical investigations are carried out as early as possible, and the recent emergence of COVID-19 precipitated a crucially urgent need to understand transmission patterns, severity, clinical features, and risk factors for infection. Effective testing can both confirm the presence of the disease in an individual and indicate the location, extent, and development of the outbreak.
Several techniques for detection and diagnosis of COVID-19 are currently under development, some of which may detect the novel virus exclusively; others may also detect strains that are genetically similar. A detection kit recently announced uses technology based on a portable lab-on-chip (LoC) platform capable of detecting, identifying, and differentiating MERS-CoV, SARS-CoV, and COVID-2019 in a single test, which integrates two molecular biological applications: polymerase chain reaction (PCR) and DNA microarray screening. Whereas traditional PCR coronavirus detection kits can take a day to produce results, the latest LoC detection kits can produce results in about two hours, and LoC technology may be the key to powerful new diagnostic instruments and point-of-care testing devices.
An LoC is a device that integrates one or several laboratory functions on a single integrated circuit. LoC devices are microelectromechanical systems (MEMS) devices (Figure 1) that function as "micro total analysis systems" (µTAS), generally using microfluidics principles to manipulate minute amounts of fluids. In practical terms, microfluidics is about doing chemistry on a tiny scale and trying to emulate nature. Biomedical microelectromechanical systems (BioMEMS) have emerged as a subset of MEMS devices for applications in biomedical research and medical microdevices, with an emphasis on mechanical parts and microfabrication technologies. Applications include disease detection, chemical monitoring, and drug delivery. There has been rapid market growth for bioMEMS technologies, and many bioMEMS devices are already commercially available; a familiar example is a blood-glucose sensor. There is also great potential for large-scale commercialization of microfluidic-based LoC technologies.
Figure 1: (a) MEMS; (b) MEMS integrated into tires for pressure sensing; (c) MEMS used as micromirrors for image projection and communications; (d) integrated MEMS.
LoC is not new. In fact, as long ago as the late 1990s, advances in microfabrication technology had enabled the development of a fully automated LoC, designed to integrate sample preparation, fluid handling, and biochemical analysis. Techniques derived from semiconductor manufacturing enabled the translation of experimental and analytical protocols into chip architectures comprising interconnected fluid reservoirs and pathways (Figure 2). By driving fluids in a controlled manner through selected pathways by electrokinetic or pressure forces, it was possible to create the functional equivalent of valves and pumps capable of performing manipulations, such as dispensing, mixing, incubation, reaction, sample partition, and detection.
The first commercially available LoC product was introduced in 1999 for the analysis of DNA and RNA biomolecules, as well as protein and cell assays, with worldwide sales of more than 7000 instruments. These LoC bioanalyzers could handle nucleic acids, proteins, and cells on the same platform using sample-specific reagents and chips and set an industry-standard for RNA analysis and sequencing. LoC for integrated chemical and biochemical analysis has also grown dramatically in the past decade. Although the primary focus has been on medical uses, the basic technology is applicable to a wide variety of analytical and monitoring functions and fits very logically into the concept of a connected world (Figure 2).
Microfluidic devices can be fabricated with a variety of materials—including glass, rigid polymers, and elastomers—using techniques such as CNC milling, injection molding, and photolithography. The original material was silicon since the fabrication techniques had been derived from semiconductor manufacture, and several alternative processes have been developed because of requirements for specific material properties, as well as lower production costs and faster prototyping. A wide variety of sophisticated chips are increasingly being demonstrated, but it is believed that few of these will be seen on the general market because of the lack of established commercial manufacturing technology. 3D printing has recently emerged as an alternative approach for the fabrication of fluidic devices and may replace soft lithography as a preferred method for rapid prototyping. But existing technologies are not unified, and it remains to be seen which processes and materials will eventually be adopted for high throughput diagnostics.
Figure 2: BioMEMS LoC. (Source: HP Laboratories, 1995)
Basic Components of an LoC
The component devices that make up an LoC are (Figure 3):
- Electrophoresis: Separation columns
- Microfluidics: Channels, valves-pumps & mixers
- Chem-bio detectors and sensors
- Microfluidic chips
Figure 3: LoC elements [1].
1. Electrophoresis
This is a method of separating large molecules (i.e., DNA fragments, blood, or other proteins) from a mixture of similar molecules by passing an electric field toward an electric pole (anode or cathode) in a liquid on various media (e.g., paper, glass, gel, liquid). It is used to separate and purify biomolecules. Each molecule travels through the medium at a different rate—depending on its electrical charge and size—and toward either the anode or the cathode at a characteristic speed (Figure 4).
2. Microfluidics
This custom application of fluidic technology is applied with conventional micromachining techniques, such as wet etching; dry etching; deep, reactive ion etching; sputtering; anodic bonding; and fusion bonding to make flow channels, flow sensors, chemical detectors, separation capillaries, mixers, filters, pumps and valves for various LoCs (Figure 4).
Flow in microchannels is laminar, which allows selective treatment of cells in microchannels, or arrays, as well as biochemical reactions. The integration of microelectronics, micromechanical, and microoptics onto the same substrates allows automated device control, which reduces human error and operation costs.
3. Chem-Bio Detectors and Sensors
Detectors, sensors, and electrodes can be ChemFET and BioFET C-MOS devices with special membranes or diffusions to make them sensitive to chemical or biological molecules. The sensors and electrodes are electrical elements that are sensitive to various chemical or biological molecules, plated with gold, silver, platinum, palladium, etc., and their salts (Figure 5).
4. Microfluidic Chips
A microfluidic chip is a set of microchannels etched or molded into a material (glass, silicon, or polymer, such as PDMS). Microchannels form the microfluidic chip connected in order to achieve the desired features (mix, pump, sort, control bio-chemical environment, etc.). Networks of microchannels are connected to the outside by inputs (inlets) and outputs (outlets) pierced through the chip (interface between the macro and micro world).
LoC Materials
Over the years, several materials have been developed for use with LoC. It started in the late 1990s with silicon, as the microelectronics industry developed various methods of micromachining silicon (MEMS) for accelerometers for airbag sensors. From silicon wafers, the materials branched out to glass and then polymers. The most recent interest has been in PCBs and the use of various paper materials.
Silicon and glass have several advantages for fabricating an LoC, while being the most expensive. Polymers and especially PCBs are a new choice because of various materials available and the integration of electronics and various printing technologies. While paper is coming into focus for research, its use is only just beginning. Table 1 lists several characteristics of each of these materials.
Table 1: Base materials for LoC formations.
1. Silicon-based
Silicon started the LoC point-of-care (PoC) diagnostic uses. Figure 6 shows one of the first on the market—the Agilent 2100 Bioanalyzer System—for DNA, RNA, serum protein, and infectious disease analysis.
Figure 6: Agilent Technology has been involved in the life sciences since 1995. Their “nanolab chips” are used to analyze DNA, RNA, SARS, and other infectious disease proteins [2].
2. Glass-based
Glass is a lower cost material if electrical components and circuitry are not required. Glass can be fabricated into microchannels and deposited with many substances such as gels and coating. The glass device seen in Figure 7 is an Agilent 3100 Bioanalyzer Automated LC/MS that comes in numerous forms to separate chemicals and biological samples into microspray streams for use with liquid chromatography/mass spectrometry (LC/MS).
3. Polymer/PCB-based
Many polymers are also optically transparent and can be integrated into systems that use optical detection techniques such as fluorescence, UV/Vis absorbance, or Raman method. Moreover, many polymers are biologically compatible, chemically inert to solvents, and electrical insulating for applications where strong electrical voltages are necessary, such as electrophoretic separation and the surface chemistry of polymers. This can also be modified for specific applications. The most common polymers used in bio-MEMS include PMMA, PDMS, OSTEmer, and SU-8.
So, what could be achieved using PCB technology? Of recent years a lab-on-printed circuit board (LoPCB) approach has been suggested. The PCB industry is mature, well-established worldwide, and has standardized fabrication processes, materials, and production equipment currently dedicated to electronics applications, but with the potential to become a natural partner for LoC development and the scope to be straightforwardly up-scaled.
Enter Dr. Despina Moschou, a researcher at the Centre for Advanced Sensor Technologies, Department of Electronic and Electrical Engineering at the University of Bath in the U.K. Dr. Moschou is a frequent speaker at printed circuit events like AltiumLive [1], EIPC Conferences, and the ICT Conferences. Fortunately, for us, she has taken the time to prepare summaries of her, and the many others in this field, work on LoC and LoPCB µTAS approaches.
Early experimentation was focused on bio-electrodes for PCBs and on the microfluidics compatible with PCB fabrication. Figure 8 shows a test vehicle. This was a two-sided FR-4 PCB with gold plated copper traces and sensor electrodes. Two different golds were tested. One was a soft gold—the Metalor R MetGold Pure ATF process, plated 2.57 µm layer of 90 HV hardness. For the hard gold, the Metalor R EnGold 2015CVR process was followed, providing 2.41 µm of gold on top of 3.41 µm of nickel with a final hardness of 140–180 HV.
To handle the delicate microfluidics, the properties of dry film photoresist (like DuPont RistonTM, or DFR) was employed. This photosensitve material, with proper curing, can be stabilized for long life, and—in some applications—can be used as a photosensitive adhesive. Too bad that the dry-film solder mask (DFSM), like DuPont VacrelTM, was no longer available. A thin FR-4 layer (200 µm) was laminated with a 50 µm DFR, that was patterned using standard PCB photolithography, developed and cured for two hours to drive off any solvents. Then, adhesive-based flexible cover coating of PMMA film was laser micromachined to provide for larger fluidic supply channels (~5 mm), and the stackup laminated to the FR-4 sensing layer.
Figure 8: The experimental LoPCB biosensing platform; (a) integrated LoPCB stackup; (b) electrochemical impedance spectroscopy electrode configuration; (c) commercially fabricated PCB biosensing platform; (d) sample delivery microfluidics [4].
Dr. Moschou’s recent work is shown in Figure 9, which is a three-layer multilayer experiment where the construction is:
- Layer 1: Reference layer plated with copper, silver, and silver chloride (Figure 5b&c)
- Layer 2: Sensing electrodes plated with hard gold
- Layer 3: the microfluidic layers for the sample solution
A 3D exploded view, as well as the plated layers, are also shown. The experimental test board proved very successful, so a full LoPCB substrate was designed (Figure 10). This fully integrated PCB cartridge (4.6 cm x 5.7 cm) includes the microfluidic channels for handling the sample, reference electrodes, and working electrodes. It is designed to measure the biomarkers for the test for tuberculosis. In addition to the three-layer construction, the cartridge contained:
- PCI express electrical interfacing
- Six channels (four standard curve points within the clinical range, one negative control, and one sample)
- 10 µL reaction chambers
- Three amperometric sensors per channel
- Full assay implemented on the PCB
Figure 9: A three-layer PCB design that includes a microfluidic on one layer, implemented in a dry-film photoresist (RistonTM), with integrated AgCl and gold electrochemical sensors [5].
This LoPCB proved to be very successful. The optical and electrochemical electrodes (sensors) provided data equal to those measured in laboratory test and in some cases, even more sensitivity—plus, there were no fluidic leaking.
Figure 11 shows the three layers of this cartridge before and after lamination. Also shown is the cartridge prepared to accept the medical fluids and human test sample. Other researchers on LoPCB have performed similar experiments and designed similar modules.
Figure 11: The three layers of the ELISA LoPCB cartridge before lamination, the finished PCB multilayer, and the LoPCB prepared for testing with biofluids and the human serum [1].
Figure 12 illustrates a portable microfluidic diluter with a variable and actively controlled dilution ratio suitable for PoC implementations. It is fabricated entirely using the developed LoPCB manufacturing technology by the same PCB manufacturers. A standard microfluidic network (Figure 12a) comprising two inlets and two outlets was designed and fabricated (Figure 12b, c, & e), where the resulting dilution ratio is thermally regulated using a power MOSFET as a heating element (Figure 12c) [6].
The manufacturing process has been developed to produce a three-layer printed circuit utilizing FR-4 laminate. The stackup utilizes a top, middle, and bottom layer. The top layer is silver plated, with the vias as pseudo reference electrodes, if pre-chlorinated. The middle layer is gold plated; thus, vias can be used as sensing electrodes since enzymes, antibodies, cells, and microorganisms can be immobilized onto the gold electrode surface, making them effective biosensors. The bottom layer serves as the microfluidic network, interconnecting the inlet and outlet vias of the PCB. The microchannels are formed in dry photoresist (Figure 13).
Figure 13: The PCB-based active control diluter stackup of the developed LoPCB; (a) exploded view; (b) cross-sectional view along the microfluidic channel [6].
Conclusion
As this technology evolves, more materials are introduced to see if the overall cost of these devices could be brought down. As seen in Table 1, paper was introduced as well as ceramics, polymers, and then PCBs. Today, 3D printing, printed electronics (PE), and various inkjet technologies are being investigated to make LoPCB cost less and more accurate for more applications.
In fact, during the month of March 2020, the U.S. Food and Drug Administration (FDA) issued emergency use authorization (EUA) to six firms to allow them to use their SARS-CoV-2 EUA test for the fastest available molecular point-of-care (PoC) test for the detection of COVID-19. These six firms are:
- Roche Holding AG (March 13) [7]: The test uses Roche's fully automated Cobas 6800 and Cobas 8800 systems. With this authorization it will have millions of tests available each month for use on the two Cobas systems.
- Thermo Fisher Scientific (March 14) [8]: The test can be run on and is optimized for use on the Applied Biosystems 7500 Fast Dx Real-Time PCR instrument. 150,000 test kits are available today, and TFS expects to ramp up to 5 million a month by April.
- Hologic Laboratories (March 16) [9]: Tests run on the automated, high-throughput molecular diagnostic platform, the Panther Fusion, which can provide results in less than three hours and process up to 1,150 coronavirus tests in 24 hours.
- Quidel (March 17) [10]: Noted that its Lyra product (Lyra SARS-CoV-2 assay for the detection of the coronavirus that causes COVID-19) line offers PCR reagent kits that can be used by laboratories equipped with molecular testing instrumentation, such as the Applied Biosystems 7500 Fast DX platforms from Thermo Fisher Scientific.
- Laboratory Corporation of America (March 23) [11]: Can perform on-site serological and molecular tests for COVID-19, using Cepheid’s recently announced GeneXpert System “Xpress SARS-CoV-2 Molecular Test” for up to 20,000 tests per day.
- Abbott Laboratories (March 27) [12]: The test runs on the m2000 RealTime Molecular System for centralized lab environments combined with ID NOW controller. It can provide positive results in five minutes and negative results in 13 minutes. Abbott is prepared to produce 50,000 test devices per day.
Other molecular PoC testing platforms from Agilent Technologies, Alere Toxicology, Acelis Health, and Sony Micronics may not be far behind.
References
- D. Moschou, “The Challenges of Redesign for Lab-on-PCB,” AltiumLive Conference, Munich, Germany, October 2018
- Agilent 2100 Bioanalyzer Product brochure.
- D. Moschou, “Commercial PCB Technology Is Advancing Point-Of-Care Medical Diagnostics,” Electronics World, June 2019.
- J. Pawan, J. Rainbow, A. Reqoutz, P. Estrela, & D. Moschou, “A PNA-based Lab-on-PCB diagnostic platform for rapid and high sensitivity DNA quantification,” Centre for Biosensors, Bioelectronics, and Biodevices, Department of Electronics and Electrical Engineering, University of Bath, U.K.
- D. Moschou & A. Tserepi, “The Lab-on-PCB approach: Tackling the uTAS commercial upscaling bottleneck,” Journal of the Royal Society of Chemistry, Vol. 17, 2017, pp. 1,388–1,405.
- N. Vasilakis, K.I. Papadimitriou, D. Evans, H. Morgan, & T. Prodromakis, “A Commercially Available Lab-on-PCB Technology for Affordable, Electronic-Based Point-of-Care Diagnostics,” Nanofabrication Centre, School of Electronics and Computer Science, University of Southampton, U.K.
- Roche, “Roche’s cobas SARS-CoV-2 Test to detect novel coronavirus receives FDA Emergency Use Authorization and is available in markets accepting the CE mark,” March 13, 2020.
- 360DX, “Thermo Fisher Coronavirus Test Gets FDA Emergency Use Authorization,” March 14, 2020.
- 360X, “FDA Grants Emergency Use Authorization for Coronavirus Assays From Hologic, LabCorp,” March 16, 2020.
- Genomeweb, “Quidel Nabs FDA Emergency Use Authorization for Coronavirus Assay,” March 17, 2020.
- Laboratory Corporation of America, “LabCorp Developing Options to Prioritize COVID-19 Testing for Inpatient Population in Support of Guidance from the White House Coronavirus Task Force,” March 23, 2020.
- Abbott, “Detect COVID-19 in as Little as 5 Minutes,” March 27, 2020.
