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104

S. Papamatthaiou and D. Moschou

fabricated by more costly advanced and sophisticated fabrication processes.

From the above-described studies, it is evident that Lab-on-PCB may be considered a promising platform for biosensing applications. It is thus logical that more effort has been focused recently on further optimisation of a PCB industrycompatible integration of the different device components. Franco etal. [36] developed a PCB compatible technique for bonding the PCB substrate to a polymeric solid material for microuidic integration. Particularly, they used PMMA but this can be easily extended to PC, polyethylene terephthalate (PET) and cyclic olen copolymer (COC). Instead of using a glue or/and adhesive tape for the bonding, they developed a thermal method as they argued that this technique was more pragmatically oriented towards mass production. A copper microheater provided the energy to temporarily melt the plastic and the bonding was completed after it solidied again. They further applied this technique on fabricating the rst reported normally open PCB-based microvalve [37]. This was done by incorporating an additional copper microheater, placed under the channel. The generated heat melted the PMMA which in return blocked the channel.

7.2.4 Advanced Quantication

Diagnostic Device Examples

7.2.4.1 PCR Modules

Adding to the proven benets of PCB implementation and the experience/knowledge regarding successful manufacturing methods, there has progress in the development of self-sufcient PCB-based diagnostic devices incorporating the previously described individual modules (e.g. mixers, pumps, etc.) with increasingly reported benchmarks matching standard, non-PCB devices or traditional benchtop methods. At Stanford University, Marshall et al. [38] integrated mixing, thermal lysis of whole blood and nucleic acid isotachophoresis extractionpurication on a single PCB chip with microuidic structure made from polyurethane. Their results were comparable to those obtained using

standard off-chip lysis and a glass capillary for ITP [39]. The validation of their on-chip lysis and extraction was performed with off-chip quantitative polymerase chain reaction (qPCR). Given the powerful relevance of amplifying specic regions of DNA by PCR for biosensor applications, miniaturisation of the underlying technology has been explored. The integration of micro PCR (μPCR) in a PCB-based device that is also capable of sample preparation and subsequent DNA detection was rst reported in 2004 by Liu etal. [40]. The device consisted of three modules (Fig.7.4b): (i) the plastic chip which included a mixing unit for cell capture using immunomagnetic separation, (ii) a cell pre-concentration/ purication/lysis/PCR unit and (iii) a DNA microarray chamber; comprising a PCB with Peltier heaters and control circuitry and a second PCB chip with 4×4 gold electrodes, where the target DNA hybridisation took place (Motorola eSensor). The plastic chip was micromachined in PC and was sealed by another (500μm thick) PC cover via solvent assistant thermal bonding. The valves were made by melting and re-solidifying parafn while the three boards were attached together by means of double-sided adhesive tape.

On a similar quest, Moschou et al. [41, 42] presented a more PCB industry compatible μPCR device, whereby the uidic compartments were made by laminating polyimide lms (Dupont PC1015) on the PCB board and the micro-heaters were made on the copper layer (Fig.7.4b). The μPCR module fed a label-free, silicon-based, capacitive DNA-sensor for mutations of the KRAS gene, of diagnostic signicance for colon cancer. Compared to earlier studies this device was simpler, requiring an external instrument for uid pumping and temperature control of the integrated micro-heaters for the μPCR steps. Subsequent development focused on deriving an efcient array of micro-heaters with a combined temperature sensing/heating feature, employing also simulations to ensure uniform temperature across each PCR zone (denaturation, extension, annealing) with no thermal cross-talk between the zones while achieving comparable DNA amplication results to commercial bench-top thermocycler in a shorter time [43]. This continu-

Pump #3

Pump #4

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105

Waste

Wash buffer

storage

Sample storage

PZT disk

Pump #1

Pump #2

Hybridization buffer storage

PCR reagents

storage

Valves

Magnet/heater

PCR

Valves

Microarray

chamber

PZT disk

PCB (yellow)

Plastic fluidic

chip

eSensor

Fig. 7.4 Integrated lab-on-PCB device. (a) Left: Schematic of the plastic uidic chip. Pumps 1–3 are electrochemical pumps, and pump 4 is a thermopneumatic pump, Right: Photograph of the integrated device that consists of a plastic uidic chip, a printed circuit board

(Reprinted from Ref. [40], copyright 2004, American Chemical Society). (b) Lab-on-PCB chip featuring μPCR and DNA-silicon sensor with laminated polyimide lms. (Reprinted from Moschou et al. [42], copyright 2013, SPIE)

(PCB), and a Motorola eSensor microarray chip.

ous ow μPCR device was further improved by the same group in terms of amplication speed and power consumption, providing a means for developing portable, battery-operated μPCR [44,

45]. The achievement of robust sealing (with-

standing 12 bars), retaining complete PCB manufacturing processes compatibility, enabled the group to increase the channel length and the ow

106

S. Papamatthaiou and D. Moschou

velocity (15 mL/min) further, consequently decreasing the amplication time to only2min, rendering it one of the fastest PCR devices in the literature regardless of the material [45].

Although the concept of creating the microuidic channels in the PCB was ideal for continuous ow μPCR applications, due to the low thermal conductivity of the material (required for consistent xed temperatures at the three individual μPCR areas), an alternative approach for static μPCR has involved microuidics in a separate PMMA formation on the PCB chip [46, 47] introducing interesting solutions (ranging from added copper layer to active fan cooling) to mitigate the higher thermal mass and the need for low thermal inertia (static PCR requires thermal cycling). Indeed, this highlights the signicant growing interest in the Lab-on-PCB approach.

Tseng et al. [48] followed a different approach for microuidic integration of a qPCR device on PCB. After constructing a threeelectrode electrochemical sensor with copper tracks as heating elements, they adhesively bonded a commercially supplied sterilised chamber on top of the board to hold the uid for the qPCR.In addition, the droplet-based microuidics on PCB could be highly suitable for DNA amplication as the copper electrodes used for electrowetting could be simultaneously used as heaters for the amplication process. This idea was recently applied to human papillomavirus (HPV) diagnosis [49].

7.2.4.2 Advanced Bio-Sensing Devices

Advancing PCB devices that specically focus on biological sensing (as shown in Table7.1), a carbon nanotube-based PCB electrode array demonstrated a state-of-the-art sensing performance, achieving simultaneous amperometric detection of lactate and glucose [50], just one of several increasingly sophisticated carbon-based sensors made possible by adopting the latest manufacturing technologies (Fig. 7.5a). Inkjetprinting was employed to deposit graphene on the working electrode of a exible PCB electrochemical sensor for wearable bio-electronics designed for continuous glucose sensing [51]. Gold nanoparticles have been electro-deposited

on graphene to enhance the sensitivity, reaching a

0.3mg/dL limit of detection (LOD). Ultrasensitive protein detection by PCB

devices has also been accomplished. Jacobs etal. [52] sputtered ZnO on PCB chips to detect a protein biomarker for cardiovascular diseases. Their aim was to exploit the inherently nano-textured ZnO surfaces for electrochemical biosensing on the cost-effective PCB platform. Screen-printed silver electrodes were made by means of conventional PCB technology to detect carcinoembryonic antigen (CEA) protein, a cancer biomarker [53]. A novel antibody-like biomimetic material has been used as a biorecognition element resulting in sensitive (pg CEA per mL), rapid (15-min maximum incubation period) and precise (5% signal change) performance at a tenth of the manufacturing cost of traditional commercial devices.

Adopting alternative approaches, the coulter

principle has been applied to enumerate tumor cells on a PCB chip [54, 55], showing comparable performance to a commercial cytometer with the added benet of PoC capability. Sanchez etal. achieved an impressive selectivity of seven breast cancer gene markers with LOD of 25 pM by multiplex amplication and detection of mRNA on gold PCB electrode-arrays [56]. Furthermore, Jolly etal. developed a DNA microuidic sensor by immobilising PNA probes on PCB gold micro-electrodes [57]. They researched two different industrially-applied PCB gold electroplating technologies (soft and hard plating), reporting LOD as low as 57 fM, highlighting signicant achievements for electrochemical DNA sensing on PCB electrodes.

Paving the way for high-quality commercial

products, Moschou et al. [58] incorporated a commercially available assay for IFN-gamma immunosensing into a double-layer PCB chip, that consisted of a reference electrode layer (silver plated) and a sensing electrode layer (gold plated). The rst layer also included cylindrical, gold-plated micro-chambers for solution handling. The described chip is shown in Fig.7.5b. This platform was later upgraded to a microuidic one, optimised for microuidic diffusion kinetics [59]. These studies were two of several assisted by partnerships between academia and

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107

(continued)

[14]/[19]

Microuidic/uid handling

technology Reference

Sensor technology (where

applicable)

Flores etal. [72]

Copper etching, PMMA Franco etal. [36]

SU-8

SU-8 Aracil etal. [66]

PDMS Cabello etal. [74]

Optical, capacitive Copper etching Wego etal. [22]

SU-8 spin coating Gassmann etal. [30]

Electrochemical

(Chronopotentiometry)

Τhermal method for PMMA-PCB

Flow sensor Thermal dispersion Spin-coated SU-8, PMMA Kontakis etal. [27]

pH-regulation system, pressure, bubble

sensor

Volume actuator, bubble detector Capacitive Copper etching Merkel etal. [20]

Table 7.1 PCB-based devices for μTAS applications

Micropump Copper etching Wego and Pagel [21]

Electrolytic pump PMMA Kim etal. [70]

Automated control of micropumps SU-8 Flores etal. [69]

Micropump Spin-coated SU-8 Luque etal. [29]

Peristaltic/diffuser, nozzle pumps Copper etching Nguyen and Huang

Application/feature Analyte (where applicable)

Bubble detector Capacitive Plastic pipes Quoc etal. [71]

Normally open microvalve Copper etching, PMMA Perdigones and Quero [37]

Single-use, unidirectional microvalve Copper etching, spin-coated

Temperature regulation Haci etal., [73]

Thermopneumatically actuated

microvalve

Emulsion generation, ow monitor Capacitive Copper etching, glass sealed Dong etal. [68]

Cell culture (microheater and

micromixer)

Passive microuidics Dry lm photoresist Vasilakis etal. [61]

Electroosmotic ow measurement Dry lm photoresist Wu etal. [15]

Droplet-based microuidics (EWOD) Gong and Kim [23]

Paddle wheel ow sensor, sensor cell

array with heaters for future sensing

bonding for microﬂuidics

applications

Microelectrode arrays (MEA) Cabello and Aracil [64]

SAW-based acoustouidics PDMS Mikhaylov etal. [67]

108

S. Papamatthaiou and D. Moschou

Alhans etal. [75]

Jobst etal. [17]

Petrou etal. [18]

Microuidic/uid handling

technology Reference

Sensor technology (where

applicable)

Dry lm photoresist, glass

cover

[51]

Dry lm photoresist/PDMS Morgan etal. [60]; Pu etal.

wafer

(Amperometric)

Pittet etal. [24]

Li etal. [50]; Kassanos

etal. 76]

PDMS Burdallo and Fern [31]

cover

Electrochemiluminescence Dry lm photoresist and glass

(Amperometric)

(Amperometric, CV and EIS)

PMMA Moschou etal. [4]

ISFET)

extended gate ISFET)

Electrochemical (ISFET) PDMS Tseng etal. [32]

Guijt etal. [35]

photoresist

Marshall etal. [38]

Polyurethane pouring with

PDMS mold and PMMA

Mavraki etal. 44]

supporting frame

Optical Dry lm photoresist Wu etal. [34]

Liu etal. [40]

Tseng etal. [48]

Machined polycarbonate

sealed by a thin PC layer

microarray)

Laminated polyimide lms Kaprou etal. [45]

chambers adhesively bonded

Electrochemical (CV) SU-8, commercial sterilised

Glucose-lactate Electrochemical

Application/feature Analyte (where applicable)

Table 7.1 (continued)

Continuous sampling Glucose Enzyme Dry lm photoresist, glass

Glucose-lactate Electrochemical

Glucose Electrochemical

Conductivity and pH Electrochemical (IDE &

Seawater thermal treatment Machined polycarbonate (PC) Gassmann etal. [25]

pH sensing Electrochemical (OCP,

Inorganic cations Electrochemical PDMS and dry lm

pH sensing for G6PD deciency

detection

Capacitively coupled contactless

conductivity detection

Lysis and isotachophoresis Nucleic acids (Malaria

detection)

On-chip mixing, thermal lysis,

KRAS gene (colon cancer) Capacitive Laminated polyimide lms Moschou etal. [41];

Esherichia coli K12 detection Electrochemical (eSensor™

Quantication of amplication

product of qPCR

Continuous ow μPCR module

Sample preparation, μPCR

PCR-compatible nucleic acid

extraction

Three electrode sensors integrated with

qPCR

Ultrafast continuous ow μPCR

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etal. [77]

Canton [49]

etal. [65]

Microuidic/uid handling

technology Reference

PMMA/Glass Kaprou etal. [47]; Shen

Plastic Diaz-Diaz and Campos-

Leiterer etal. [63]; Luo

etal. [79]

and pressure sensitive

adhesive sealing/PDMS

Dry lm photoresist Vasilakis etal. [80]

109

PDMS Ghanim etal. [81]

PMMA Moschou etal. [58]

PMMA Acero Sánchez etal. [56]

Sensor technology (where

applicable)

Application/feature Analyte (where applicable)

Static μPCR

Electrochemical (EIS) PDMS Narakathu etal. [78]; Ren

Metal compounds/circulating

tumor cell

Droplet microuidics for isothermal

DNA amplication

(Amperometric)

Tumor cell Electrochemical (impedance) PDMS Guo etal. [13]

Enumeration of tumor cells Electrochemical (impedance) PDMS Shi etal. [54; Fu etal. 55]

Dielectrophoresis-based cell sorting Copper etching, olen-sheet

Microuidic active control diluter Tetramethybenzadine (TMB) Electrochemical

Circular diaphragm resonator Machined PMMA Ortiz etal. [26]

CEA antigen/HPV DNA target

recognition

DNA separation & detection Electrochemical

(Amperometric)

Electrochemical

(Amperometric)

IFN-γ sensor for tuberculosis

Electrochemical (EIS) PDMS Jacobs etal. [52]

DNA Electrochemical (EIS) PMMA sealed by thin FR-4 Jolly etal. [57]

Serum protein–PAH adducts Hall effect Glass Wu etal. [28]

Protein biomarker for

Electrochemical (DPV) Moreira etal. [53]

cardiovascular diseases

(Troponin-T)

CEA protein (cancer

biomarker)

mRNA Electrochemical

(Amperometric)

110

S. Papamatthaiou and D. Moschou

Fig. 7.5 Examples of integrated microuidic channel applications on PCB. (a) Photograph of the array chip used for multi-biosensors base. The four gold rings are to be modied to work as Ag/AgCl reference electrodes, the four larger disk electrodes within the ring electrodes work as counter electrodes, the 16 smaller disk electrodes are to be fabricated as lactate, glucose sensors and sensor layers without enzyme as interference detection sensors. (Reprinted from Ref. [50], copyright 2013, Elsevier publisher). (b) Commercially fabricated micro-chambers

the PCB industry [4, 33, 47, 60–62] showing the up-scaling potential of the PCB platform for LoC applications.

PCB devices in the LoC eld have also been purposed for dielectrophoresis using nickel and gold plated electrodes [63] (Fig.7.5c), 3D microelectrode arrays (MEA) for the detection of electrical signals from cells or tissues [64], electrical impedance spectroscopy (EIS) for tumor cells detection [65], thermopneumatical actuation of single use microvalve [66] and a surface acoustic wave (SAW)-based acoustouidic PCB device [67]. A particularly interesting application of integrated microuidic channels on the PCB (although sealed with a glass chip) was devel-

used for IFN-γ detection. (Reprinted from Ref. [58], copyright 2016, Elsevier publisher). (c) PCB-chips for dielectrophoresis. Circular and square-shaped copper electrodes plated with nickel and gold to minimise electrochemical oxidation on the electrodes. (Reprinted from Ref. [63], copyright 2015, SPIE). (d) 3D schematics of the USB-driven microuidic device on a PCB (U-Chip). The inset shows the fabricated U-chip on a PCB with a standard USB interface. (Reprinted from Dong et al., [68], copyright 2013, Royal Society of Chemistry publisher)

oped by Li etal. [68] whereby the copper electrodes were used to generate oil-water emulsions by electrolysis (Fig.7.5d).

7.2.5 Recent Developments inLabon- PCB Commercially Relevant Issues

The engagement of the PCB platform for use as an integral part of biosensing applications, advancing the Lab-on-PCB concept, was evidenced by detailed studies that focused on PCB material characterisation and quality control of properties, aiming to render more reliable and

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111

Fig. 7.6 Computer assisted design software facilitates microuidic design. A passive micro-mixer component and its inlet designed in Altium® and a microuidic footprint library that includes various components

robust sensors, suited for commercial use. Stable PCB Ag/AgCl electrodes could serve as biosensor integral reference electrodes [4, 82] and longterm hydrophilisation stability of FR-4 would be advantageous for passive microuidics [61, 83]. More specically, the PCB reference electrodes demonstrated stable open circuit potential behavior under continuous buffer ow of various pH values and the suitably treated FR-4 surfaces

ing in improved communication with the factory and unhindered implementation of the design during the manufacturing phase. Key steps in the realisation of this idea involve the layer stack manager conguration and the design rule check (DRC) set of the CAD software to facilitate the microuidics design along with the creation of libraries dedicated solely to the microuidic and

sensing components (Fig.7.6). retained their hydrophilic properties for at least 26days.

Furthermore, unication of electronic and

7.3 Conclusion

microuidic manufacturing processes in the PCB industry mandates the same unifying practice in the design phase. Hence, adoption of the PCB industry standard CAD software to design the microuidic structures of the Lab-on-PCB platform represents a very welcome recent ambition [84, 85]. Essentially, this achieves merger of electronic and microuidic design within a single computer assisted design (CAD) platform result-

An extensive overview of PCB-based LoC proto-

type development by the research community,

reveals an evolution from mostly individual com-

ponents present in pioneering devices, to two

decades of enhanced integration, establishing

more complex and self-sufcient platforms serv-

ing the μTAS approach. The Lab-on-PCB plat-

form can successfully accommodate most

112

S. Papamatthaiou and D. Moschou

diagnostic related application, enabling highly accurate analyte quantication at the point of need, ideal characteristics for potency assay applications. Highly advantageous features of Lab-on-PCB technology are the long-standing industrial infrastructure, established appropriate micro-fabrication capabilities and the intuitive electronics integration. Research focus on manufacturing techniques and materials for integration of microuidics with mature and established PCB industry practices, has achieved successful proofs of principle in recent years. In several cases, the microuidic integration is accomplished by bonding the uidic compartment, usually made from glass/PMMA/PDMS, onto the PCB chip housing the electronics. Alternatively, channel formation directly on the PCB, i.e. by metal etching, essentially using the metallic layer’s thickness as the channel’s walls, introduces a holistic integration simplicity that distinguishes of the Lab-on-PCB LoC eld. These hallmarks lay foundation for the full exploitation of the upscaling advantages that the PCB platform offers with promise for cost-effective potency assays. Use of graphene ink drop-casted to form a transistor channel helped establish the rst example of an electrolyte gated eld-effect transistor (FET)-based PCB biosensor [86] introduces versatility for sensitive measurement of a broad range of potential biomarker types [87].

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