Sustainable Materials and Technologies 25 (2020) e00205
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Sustainable Materials and Technologies
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Laser induced graphene for biosensors
Zhengfen Wan a,b, Nam-Trung Nguyen a, Yongsheng Gao b,c, Qin Li a,b,⁎a Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan, QLD 4111, Australiab School of Engineering and Built Environment, Griffith University, Nathan, QLD 4111, Australiac Institute for Integrated and Intelligent Systems, Griffith University, QLD 4111, Australia
Article history:Received 28 May 2020Received in revised form 20 July 2020Accepted 22 July 2020Available online xxxx
Biosensors can sensitively and selectively detect a wide range of compounds and macromolecules strongly rele-vant to human health diagnosis and environmentmonitoring. Laser induced graphene (LIG) fabricated from pol-yimide has recently received intense interest for biosensor application due to its unique properties, such as three-dimensional macroporous structure, good conductivity and superior facile laser fabrication process. This laser di-rect writing technology demonstrates a great potential for developing graphene-based electronics for itschemical-free and direct patterning of graphene, as well as suitability for roll-to-roll production. In this review,we summarize the recent development of the fabrication of LIG and itsmodification formeeting the needs of bio-sensor development. The LIG has been directly employed as electrode, modified with enzyme, aptamer or othercatalyst for biosensing. The review also highlights integrated LIG biosensors that can simultaneously measuremultiple objectives.
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1. Introduction
Biosensors are analytical devices that convert a biochemical/biolog-ical reaction into ameasurable physico-chemical signal, which providesa quantitative assessment of the analyte concentration [1]. Biosensorscan detect a wide range of chemicals with applications in food security,diseases analyses, and environmental safety [2]. Recently, graphene hasattracted significant interests for biosensor applications such as glucosemonitoring [3,4], DNA sensing [5,6], and folic acid detecting [7,8], owingto the extraordinary electrical and optical properties of the graphene.Reliable, scalable fabrication and modification methods for developingbiosensors are highly on demand to bridge the existing gap betweenlab research and commercialisation [9].
Many approaches have been developed to produce graphene such asmechanical exfoliation [10], chemical vapor deposition (CVD) [11] andthe reduction of graphene oxide [12–14]. In 2014, Tour et al. fabricatedmacroporous laser induced graphene (LIG) from commercial polyimide(PI) films with a CO2 laser, exhibiting high electrical conductivity andlarge specific surface area. This laser based reduction method forgraphene shows unique advantages of selective and localized reduction,flexibility in patterning and no requirement for chemicals [15]. Moreimportantly, the precursor of PI and the effective laser treatment dem-onstrate great potential for roll-to-rollmanufacturing [16],which is crit-ical for future commercial applications of LIG-based devices. Followingworks have demonstrated the potential PI-derived LIG in various appli-cations, including nitrogen sensor [17], strain sensor [18,19], gas sensor[20,21], and acoustic sensor [22], antibacterial application [23,24], en-ergy storage [25,26], and antennas [27] etc. Compared to graphene fab-ricated with other method, LIG generates a number of defects, which isnot always a disadvantage. Li et al. and Banerjee et al. demonstrated thatelectron transfer kinetics are tremendously enhanced at graphene edgesites due to its abundance in corrugations and defect densities [28,29].The remaining oxygen components of LIG though decrease its conduc-tivity, likely weakening the signal strength of graphene biosensor,they improve its hydrophilicity and provide active sites for functionali-zation, which are critical for biosensors. Moreover, the porous 3D net-work structure of LIG can improve the efficiency in charge transferandmass transport due to its large specific surface area and highly con-ductive pathways [30–32]. Hence, LIG with its 3D architecture enrichedwith edge planes without complex processing and additive approach isa highly promising material for biosensor [33,34].
In this review,we summarize the recent developments of biosensorsbased on LIG.We pay particular attention to themodification of LIG andthe diverse biosensing applications of this technology. Current chal-lenges and future advancement, such as the integrated flexible devicesare discussed. In these studies, various precursors, mainly commercialpolyimides, were employed for laser reduction. After laser irradiation,the resulting graphene materials are referred differently in papers aslaser-induced graphene [35], laser-scribed graphene [36], or laser car-bonized nanomaterials [16]. The term ‘laser induced graphene (LIG)’ isadopted consistently throughout this paper.
2. Laser induced graphene
2.1. Fabrication of laser induced graphene
In 2014, Tour's group reported porous graphene films fabricationfrom commercial polymer films using a CO2 laser [37]. The LIG exhibitshigh surface area (≈340m2 g−1), high thermal stability (>900 °C), andexcellent conductivity (5–25 S cm−1) [38]. Fig. 1(a-c) shows the laserpatterning process on PI, SEM images and Raman spectrum. The 3D po-rous structure, which renders enhanced-accessible surface areas and fa-cilitates electrolyte penetration into the active materials, can be formedby the release of gas produced during laser treatment. The LIG demon-strates a 2D Raman band (centered at 2700 cm−1), which is typicallyfound in 2D graphite consisting of randomly stacked graphene layers
along the c axis [39]. Considering the long wavelength (10.6 μm) oflaser, the LIG formation is more likely to be caused by photothermal ef-fects. The extremely high localized temperatures (>2500 °C) induced bylaser irradiation could easily break the bonds including C−O, C_O andC − N bonds. Simultaneously, the aromatic compounds are thenrearranged to form graphitic structures.
Diverse laser with varied wavelength, such as CO2 laser (10.6 μm)[16], semiconductor laser (405 nm) [40], and near-infrared (NIR) laser(1064 nm) [41] were reported to fabricate LIG. The mechanism oflaser reduction largely depends on the wavelength of lasers, which de-termines the photochemical effect and photothermal effect involvedin the reduction process induced by laser [42]. Two sub-processes,namely the direct conversion from sp3 to sp2 carbon and removal of ox-ygen functional groups coexist during the laser reduction process [15].Diverse substrates such as cloth, paper, potato skins, coconut shells,cork [43], wood [44], lignin [45], and phenolic resin [46] can be trans-formed into graphene directly by laser irradiation, Fig. 1(d-g). Fig. 1(h) illustrates the scheme for industrial production of LIG film and itsmodification based on the precursor of commercial polymer and the ef-fective laser reduction process. Compared with other graphene sensor,this LIG sensor shows the great advantage of facile fabrication and thehuge potential for commercialization. The LIG sensor can be patternedby direct laser writingwithout additional chemical or high temperaturetreatment. The PI precursor is ready for roll-to-roll production and laserprocess is effective and applicable for large-scale production. The laserdirect writingmethod is also considered to be an effective for graphenefabrication. Kaner et al. reported that more than 100 graphene devicescan be produced over large areas in 30 min or less by direct laser writ-ing, demonstrating scalable fabrication of graphene [47]. The combina-tion of commercial precursor such as polyimide, an effective laserdirect writing system, and an automated roll-to-roll fabrication processholds the key tomaking LIG an industrialized production for biosensors.
2.2. Modification of laser induced graphene
Themodification of LIG with heteroatom doping or the formation ofa composite has been employed as an effective approach for the engi-neering of properties and performance [38]. Three strategies havebeen reported as follow.
1) Laser parameters adjustment, atmosphere and process.
Variousmethods have been developed to tune or improve the phys-ical and chemical properties of LIG by varying the laser parameters orcontrolling the reduction atmosphere, Fig. 2(a-c). Yang et al. demon-strated the surface modification of LIG, including surface morphologies,carbonization and wettability by adjusting laser powers, scanningspeeds and pulse repetition frequencies [16,49]. Controlling the laser re-duction atmosphere (O2, air, Ar, H2, and SF6) can modulate the watercontact angle of LIG from 0° (O2 or air) to >150° (Ar or H2) or > 160°(SF6) [50]. The structure of PI-derived LIG can bemodified from its orig-inal macroporous foam to an intermediate concave corrugated tilestructure and to a final carbon nanotube structure by repeated laser ir-radiations [51]. The abundant hydrophilic surface of LIG facilitates largeamounts of defects and accessible oxygen containing functional groups,which can provide numerous active sites and accelerate electron trans-fer between the electrode and species in solution [52]. By modified theproperties of LIG, the combined effects of the specific surface area, hy-drophilic surface, electronic conductivity and available edge planesites can adjusted be to enhance the electron transfer rate and analyticalperformance in LIG biosensors [53,54].
2) Doping of LIG.
Tour's group fabricated boron-doped LIG from H3BO3 mixed poly(amic acid) (PAA)with a CO2 laser (Fig. 2(d)), and themicrosupercapa-citors based on the boron doped LIG exhibit a high areal capacitance
Fig. 1. (a) Schematic illustrate of laser patterning on PI. (b) SEM image of LIG on the PI substrate, scale bar 10 μm. Inset is the corresponding SEM imagewith highermagnification, scale bar1 μm. (c) Raman spectrum of a LIG film and the starting PI film. Reproduced with permission [37]. Copyright 2014, Nature Publishing Group. (d) The letter “R” in LIG induced from bread.(e) Potato scribed with the laser to form LIG in the “R” pattern (2 cm tall). (f) LIG on gray muslin cloth in the shape of an owl. (g) LIG on muslin cloth wrapped around a marker. All owlsdepicted are 60mm in height. Reproducedwith permission [43]. Copyright 2018, American Chemical Society. (h) Scheme for roll-to-roll production of LIG film. LIG isfirst formed on the PIsheet, and then developed in a catalyst bath to form hybrid material. Reproduced with permission [48]. Copyright 2018, American Chemical Society.
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of 16.5 mF/cm2 (3 times higher than nondoped devices) and an 5–10times increased energy density [55]. In a similar process, Co3O4, MoO2,or Fe3O4 were individually mixed into PAA solution for laser treatment,contributing to the metal nanocrystals embedded LIG, which exhibit
excellent oxygen reduction reaction (ORR) catalytic activity [56]. Het-eroatom doping in carbon material demonstrate great potential toimprove the electrocatalytic activity in biosensor [57,58]. Comparedto ordinary graphene materials, the N-doped graphene provides
Fig. 2. (a) Schemes for the fabrication of LIG inside a controlled atmosphere chamber. (b) The relationship between contact angle (bars), D/G ratio (black dots), and 2D/G ratio (whitecircles) for LIG samples made under different gas atmospheres. Reproduced with permission [50]. Copyright 2017, John Wiley and Sons. (c) Sheet conductivity of LIG trace onpolyimide as a function of laser fabrication parameters (power and speed). Reproduced with permission [16]. Copyright 2015, American Chemical Society. (d) Synthetic scheme for thepreparation of boron doped LIG and fabrication of the boron doped LIGmicrosupercapacitor. Reproduced with permission [55]. Copyright 2015, American Chemical Society. (e) The TEMimages of LIG embedded with MoO2. The inset shows the high-resolution TEM images showing crystalline MoO2 in LIG. Reproduced with permission [56]. Copyright 2015, AmericanChemical Society. (f) SEM images of the LIG structures with electroless plated Ni. Reproduced with permission [40]. Copyright 2011, Elsevier. (g) Cross-sectional SEM images of LIGwith electrodeposited MnO2. Scale bar is 100 μm. Reproduced with permission [61]. Copyright 2015, JohnWiley and Sons.
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significantly enhanced oxidation currents for the enzymatic detection ofglucose [59]. Metal nanoparticles (Au, Pt, etc.) doped graphene also ex-hibited excellent electrocatalytical activity for biosensing, resultingfrom the large surface area and good electrical conductivity of graphene,and the synergistic effect of graphene and metal nanoparticles [60].Therefore, in-situ doping and metal nanoparticle doping of LIG can beeffective strategies to enhance the performance of LIG biosensor.
3) Post-surface modification (Fig. 2(e-g)).
Pseudocapacitive materials such asmanganese dioxide (MnO2), fer-ric oxyhydroxide (FeOOH) or polyaniline (PANI)were simply electrode-posited on the surface of LIG for all-solid-state flexible supercapacitorswith improved performance [61]. By using a laser direct writing tech-nique and an electroless Ni plating, the LIG/Ni composite structureswere prepared with a low resistance of less than 0.1 Ohm/sq., demon-strating an integrated wireless charging and storage device and anear-field communication (NFC) tag applications [40]. With post-
surface processing, such as, deposition, dispersing, polymerization, orbinding, single strand DNA (ssDNA), RNA, antibodies, enzyme mole-cules, and aptamers can be immobilized onto LIG surface for direct tar-get probing [62,63].
The LIG demonstrates a great potential for developing environmen-tally friendly electronic devices. In terms of the rawmaterial, besides thecommercial PI that shows stable thermal, chemical and mechanicalproperties. Various natural precursors such as wood, cotton, paper andlignin (an abundant natural polymer in plants) were extended for thefabrication of LIG [43,44,64]. In terms of the laser irradiation process,the fabrication of LIG and device patterning can be simultaneously com-pleted without traditional etching, lithography and deposition process.There is no involvement of strong acid and oxidants, which can causepollution or high temperature furnaces that requires enormous con-sumption of energy [15]. The thickness of LIG can be modulated from20 μm to 38 μm on the 125 μm PI by different laser parameters [65].The repeated laser irradiations on PI-derived LIG can modify the mor-phology and structure of LIG [51], indicating the possible recycle of
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the LIG products. In terms of device performance, LIG applications re-flect distinguished electrical and mechanical properties and demon-strate excellent stability and cyclability [21,61], which illustratepossible future application in wearable and flexible electronics. There-fore, it is very promising that the further development of natural precur-sors, the simple and repeatable laser process and the high performanceof LIG devices contribute to environmentally friendly andbiocompatibleelectronic applications.
3. Laser induced graphene for biosensor
3.1. LIG-based sensors for ionic species
Li et al. reported a simple one-step and scalable direct-laser-writingmethod for the LIG in situ decorated with metal sulfide (MS) nanocom-posite (CdS and PbS) as an efficient transducer for photoelectrochemicaldetection of Cu2+ with low detection limit of 2.0 nM and good selectiv-ity shown, Fig. 3(a-c) [66]. The LIG decoratedwithmetal nanocompositephotoelectrode were synthesized by laser irradiation on the metal-complex containing polyethersulfone on indium tin oxide (ITO) glass,simultaneously producing the LIG and the crystallization of laser-induced metal sulfide [66]. The LIG with CdS interact with Cu2+, gener-ating CuxS (x= 1, 2), which serves as exciton trapping sites on the sur-face, decreasing the efficiency of charge separation and contributing to aprominent decline of photocurrent output in LIG with CdS photoanode[67].
Glaussen et al. demonstrated the LIG functionalized with a poly(vinyl chloride) (PVC)-based ion-selective membrane for electrochem-ical sensing of plant available nitrogen (NH4+ andNO3−) in soil samples(Fig. 3(d-e)) [17]. This LIG solid contact ion-selective electrodes (SC-ISE) SC-ISEs demonstrates near Nernstian sensitivities (51.7 ±7.8mV/dec forNH4+ and− 54.8±2.5mV/dec forNO3−), lowdetectionlimits (28.2 ± 25.0 μM for NH4+ and 20.6 ± 14.8 μM for NO3−), and alinear sensing range of 10−5–10−1 M for both. With a facile low-costfabrication process and no requirement ofmetallic nanoparticle decora-tion, LIG electrochemical sensors are promising for a wide range of in-field or point-of-service applications for soil health management. In asimilar way, Kucherenko et al. reported LIG electrodes functionalizedwith ion-selective membranes for monitoring the concentrations ofNH4+ and K+ in urine samples [68]. This electrochemical LIG sensor ex-hibit a broad sensing range (0.1–150mM for NH4+ and 0.3–150mM forK+) with high stability across a wide pH range (3.5 to 9.0) in aqueoussolutions [68].
3.2. LIG-based sensors for small molecules
3.2.1. Ascorbic acid (AA), dopamine (DA) and uric acid (UA)Dopamine (DA) is an important neurotransmitter, which plays a sig-
nificant role in the central nervous, renal, hormonal, and cardiovascularsystems [69]. Its detection has gained significant attention. Due to theoverlapping voltammetric response between DA and its coexistingascorbic acid (AA) and uric acid (UA), it is a challenge to distinguishthe coexistence of DA, AA, and UA in a biological environment [34]. Xuet al. applied a poly(3,4-ethylenedioxythiophene) (PEDOT) modifiedLIG for a highly selective electrochemical dopamine (DA) sensor inthe presence of ascorbic acid (AA) and uric acid (UA) with high se-lectivity, sensitivity of 0.22 ± 0.01 μA/μM and a low detection limitof 0.33 μΜ [70]. PEDOT, a conducting polymer was electrodepositedon the LIG electrodes to promote electron transfer responses inelectrochemical sensors due to its high electrical conductivity,high stability and excellent biocompatibility [71,72]. The PEDOT-LIG electrodes demonstrate great potential for biosensors, as wellas other integrated bioelectronic devices. Nayak et al. reported Ptnanoparticles decorated LIG for electrochemical sensors that can si-multaneously detect biomarkers including AA, DA and UA with highsensitivity of 250.69 μA mM−1 cm−2 (AA), 6995.6 μA mM−1 cm−2
(DA) and 8289 μA mM−1 cm−2 (UA) and selectivity in a wide con-centration range, Fig. 4 [33]. The 3D macro/mesoporous LIG withabundant edge plane sites and large electrochemical surface area,facilitates ion diffusion and leads to efficient electron transfer. ThePt nanoparticles were then simply electrodeposited on the surfaceof LIG to improve electron transfer and sensitivity of the sensor.
3.2.2. Hydrogen peroxide (H2O2)Hydrogen peroxide, one of the most commonmolecules in biolog-
ical tissue is a general enzymatic product of oxidases and a substrateof peroxidases [73,74]. Hydrogen peroxide plays an important rolein physiological processes such as diabetes, lung disease, cancer,neurodegenaration and aging [75]. Therefore, it is of great interest todevelop an effective, easy, real-time and accurate H2O2 detectiontechnology. Nanoparticles decoration on the surface of graphene hasbeen reported as an effective approach for non-enzymatic H2O2 detec-tion [76]. Zhang et al. reported a simple and low cost non-enzymaticelectrochemical detection of hydrogen peroxide (H2O2) with Pt deco-rated LIG electrode (Pt/LIG), which exhibited an improved electro-chemical performance with a limit of detection of 0.1 mM and asensitivity of 248.4 mA mM−1 cm−2 [77].
3.2.3. UreaUrea measurement has been attracting great attention due to its re-
lations with the health hazards such as kidney dysfunction [78], cardio-vascular events [79], and environment events [80,81]. Sharma et al.fabricated a pH-based, flexible and catheter-compatible urea sensorbased on modified LIG, which can successfully detect urea concentra-tions as low as 10−4 M with a response time of less than one minute,Fig. 5(a) [82]. By electrodepositing the chitosan hydrogel films on thesurface of LIG, the quantity of the immobilized urease enzyme can beimproved due to the electrostatic and covalent immobilization strate-gies of chitosan. These urease enzymes can catalyze the hydrolysis ofurea into carbon dioxide and ammonia [83], which simply can be de-tected with a direct pH indicator paper. The low-cost materials and in-struments make this technique attractive to a large range of potentialusers.
3.2.4. ThrombinFenzl et al. demonstrated a universal modification route of LIG
electrodes for electrochemical biosensor as shown in Fig. 5(b). Thesensor exhibits a low thrombin detection limits of 1 pM in bufferand 5 pM in the complex matrix of serum [84]. After fabricatingLIG with a CO2 laser on commercial PI, the 1-pyrenebutyric acid(PBA) was immobilized to the surface of LIG via π-stacking and hy-drophobic interactions and then amino-functionalized bioreceptorswere conjugated to the carboxyl groups of PBA using standard cou-pling chemistry [84]. Avoiding the existing complex sensors andmethods, this modification strategy is an effective approach for de-veloping sensitive, flexible and miniaturized LIG sensors for ad-vanced clinical diagnostics.
3.2.5. Bisphenol ABisphenol A (BPA) is a monomer broadly used in diverse consumer
products and its concentration in water is significant for evaluatingwater quality and risk levels of human health and environment[85,86]. Hu et al. presented a facile method to fabricate BPA sensorswith a limit of detection of 58.28 aM and a response time of 20 s onthe flexible PI by dual-beam direct laser writing, demonstrating an ef-fective approach for low-cost practical sensing for on-site environmen-tal monitoring (Fig. 5b-c) [87]. A 1030-nm femtosecond (fs) laser(400 fs, 120 kHz) and a 532 nm continuouswave (CW) laser were com-bined for the fabrication of the LIG, integrating both themultiphoton ef-fect and the thermal effect of laser [88], to irradiate a commercialflexible PI sheet under ambient conditions. After plasma treatment,the LIG electrode were functionalized with receptor incubation and
Fig. 3. (a) Schematic illustration for the laser treatment of the Cd2+-containing polyethersulfone (PES) on indium−tin oxide (ITO), (LI-CdS-G@ITO) photoelectrode. (b) The photograph ofCd2+-PES on ITO (left) and LI-CdS-G@ ITO (right). (c) Principle of the Photoelectrochemical (PEC) Cu2+ sensor on LI-CdS-G@ITO photoanode. Reproducedwith permission [66]. Copyright2019, JohnWiley and Sons. (d) Photographoffive LIG solid contact ion-selective electrodes (SC-ISEs) on a single polyimide swatch and the illustration of SC-ISE ion sensing. (e) Soil columnstudies with LIG SC-ISEs compared to commercial ion probes. Representative real time plots of NH4+ and NO3− ions during soil column flush experiments. Reproduced with permission[17]. Copyright 2018, American Chemical Society.
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uncovered surface blocking process, which minimized the non-specificbinding [89]. Capacitive sensing based on AC electroosmotic (ACEO)was applied to detect the binding of BPA with aptamer on the electrodesurface, since the variation of the interface properties can trigger theshift of the interfacial capacitance [87].
3.2.6. GlucoseTehrani et al. developed a sensitive, enzyme-less glucose sensor
based on LIG decorated with copper nanocubes (CuNCs). The sensorshows a linear behavior in the range of 0.25 μM - 4 mM, an excellentsensitivity of 4532.2 μA mM−1.cm2 and a low detection limit of250 nM as well as a rapid amperometric response of less than 3s [90].This sensor demonstrates great potential for glucose detection in tears,saliva, sweat, and partial in urine. The porous LIG with an abundanceof crystallographic defects and large surface area enhances theelectroplating process of the CuNCs (as the catalyst for oxidation of glu-cose) and increases loading of the highly reactive CuNCs as well as ac-cessibility of glucose molecules. The oxidation of glucose at the CuNCsmodified electrode may occur in the following process: (1) the CuNCswere oxidized into CuO in the alkaline media; (2) CuO is electrochemi-cally oxidized to Cu(III) species; and (3) glucose is irreversibly oxidizedby the Cu(III) species [91,92].
3.2.7. Biogenic aminesAs an indicator of food safety and quality, biogenic amines (BA) is
the product of microbial metabolism in foods and its level is affectedby temperatures and storage conditions [93,94]. Vanegas et al. reporteda low-cost LIG biosensor for BA detection, which exhibits an averagehistamine sensitivity of 23.3 μA mM−1 with a low detection limit of11.6 μM and a response time of 7.3 s [95]. Following electroplating ofthe copper nanocubes, the modified LIG electrodes were thenbiofunctionalizedwith diamine oxidase (DAO) to provide selectivity to-ward biogenic amines since DAO enzyme can catalytically deaminatethe primary amines, diamines, and substituted amines to aldehyde, am-monia, andhydrogenperoxide [34], which are then readily decomposedat a working electrode polarized at +500mV and produce an oxidativecurrent [95]. The decorated LIG biosensors were successfully used tomeasure the total BA concentration in fish paste samples subjected tofermentation with lactic acid bacteria, demonstrating its great potentialfor screening food samples, diminishing food waste and reducing thechance of foodborne disease outbreaks [95].
3.3. LIG-based sensors for nucleic acids
MicroRNAs (miRNAs, ~22 nucleotides), small noncoding single-stranded RNA molecules play regulatory roles in cell development and
Fig. 4. Schematic illustration of the fabrication of LIG and Pt/LIG electrode patterns on PI sheet. (a) Fabrication of arrays of electrodes on PI sheet by laser scribing. (b) 3D view of the LIGelectrodepattern. The projection displays vertical cross-sectional SEM image of a laser-scribed PI sheet showing porous andprotrudedmorphologyof graphene. (c) Selective passivation ofelectrode area by a PDMS. (d) Electrodeposition of Pt nanoparticles selectively on the working electrode area. The projection displays SEM image of homogeneously anchored Ptnanoparticles over graphene sheets. (e) Digital photograph of patterned electrode arrays on PI sheet. (f-k) Differential pulse voltammetry (DPV) at LIG at different concentrations of(f) AA, (g) DA, and (h) UA in 0.1 M PBS (pH 7.0) as supporting electrolyte. DPV at Pt/LIG for various concentrations of (i) AA in a mixture of 4 × 10 −6 M DA and 4 × 10 −6 M UA,(j) DA in a mixture of 30 × 10 −6 M AA and 4 × 10 −6 M UA, (k) UA in a mixture of 40 × 10 −6 M AA and 4 × 10 −6 M DA, Insets: plots of the oxidation peak current density versusconcentration of each biomarker. Linear fitting is used to determine the sensitivity of the electrodes for each biomarker. Reproduced with permission [33]. Copyright 2016, John Wileyand Sons.
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are significant biomarkers for cancer and other diseases [96,97]. We re-cently reported a sensitive LIG biosensor for femtomolar microRNA(miRNA) detection shown in Fig. 6(a-c) [65]. With laser treatment onPI, the LIG with an effective N-doping (2.4% - 4.5%) contributes to thelow resistance of LIG and likely improves the affinity with nucleicacids. This self‑nitrogen-doped porous LIG was patterned as an
electrode for electrochemical sensing. Combined with the miRNA ex-traction and magnetic isolation process, specific purified miRNAs weredirectly adsorbed on the surface of LIG electrode and then electrochem-ically quantified. The detection ofmiRNAup to a concentration as low as10 f. with excellent reproducibility can be achieved, indicating the greatpotential for miRNA detection in biomedical applications.
Fig. 5. (a) Schematic diagram of enzyme adsorption on LIG with and without chitosan layer. Reproduced under the terms of the Creative Commons Attribution 4.0 International license[82]. Copyright 2019, Nature Publishing Group. (b) Schematic of the LIG Electrode fabrication, the functionalization process and the electrochemical thrombin detection mechanism.Reproducedwith permission [84]. Copyright 2017, American Chemical Society. (c) Schematic of ultrasensitive LIG capacitive sensorswith directedmovement of complex sample particleswith the applied ACEO effect. (d) Detection response of BPA samples as a function of concentration. Reproduced with permission [87]. Copyright 2016, American Chemical Society.
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Fig. 6. The schematic of the LIG biosensor for miRNA detection. (a) The fabrication process of LIG biosensor, the image of the laser written electrode (left), the Raman spectra of the LIG(middle) and the SEM image of the LIG with scale bar 1 μm (right). (b) The absorption process of the purified target miRNA onto the surface of LIG electrode. (c) The electrochemicaltest with LIG working electrode. The typical differential pulse voltammetric(DPV) signals of the LIG adsorbed with RNA decreases in comparison to the bare LIG electrode. Reproducedwith permission [65]. Copyright 2020, Elsevier.
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3.4. LIG-based sensors for immunology
3.4.1. ChloramphenicolCardoso et al. reported a LIG electrochemical biosensor with
molecularly-imprintedmaterial as biorecognition element for detectingchloramphenicol (CAP), which is an antibiotic effective against manybacteria and a commonly found contaminant in the aquatic environ-ment, Fig. 7(a-c) [98]. The biorecognition elements were assembledon the surface of LIG with molecularly-imprinted polymer (MIP) tech-nology as the following procedures: (1) electrochemically polymeriza-tion of the 3,4-ethylenedioxythiophene (EDOT) in the electrodesensing area to render a homogeneous surface; (2) incubation in0.01 M 4-aminothiophenol (4-AMP) to ensure the bonding betweenPEDOT and the MIP layer; (3) electropolymerization of functionalmonomers eriochrome black T (EBT) in the presence of the templateCAP; (4) the removal of CAP template by incubating the sensing layerin acetonitrile (CAN) and performing consecutive cyclic voltammetry(CV) scanning [98]. This LIG biosensor exhibits the linear responsefrom 1 nM to 10 mM, a high sensitivity with a limit of detection of0.62 nM and good selectivity, representing a promising approach forthe future of on-site analysis in different contexts, including environ-ment, industrial and health applications [98].
3.4.2. Acetylcholinesterase inhibitorA reliable and sensitive detection of the acetylcholinesterase (AChE)
inhibitor is highly on demanded because the inhibition of AChE contrib-utes to the continuously stimulation of the nerve conduction [99]. Geet al. report a rapid and stable photoelectrochemical (PEC) enzymaticbiosensor based on a TiO2 decorated LIG on ITO glass, demonstratingsensitive detection of an AChE inhibitor (15.4 pM) shown in Fig. 7(d-e) [99]. Considering that the graphene/TiO2 composites can extend thelight absorption range and improve efficiency of electron–hole pair dis-association and electron collection [100,101], the TiO2-decorated LIGphotoelectrodes were fabricated by laser treatment of Ti4+−containingpoly (amic acid) (Ti4+-PAA) on the surface of an ITO. The photocurrentoutput can be significantly enhanced by incubating acetylthiocholine(ATCl)withAChEbecause theAChE can catalyzehydrolysis of ATCl, pro-ducing thiocholine as an ideal hole scavenger [102]. Conversely, an ob-vious photocurrent decrease is noticed when the AChE inhibitor was
involved to impaired enzymatic activity of AChE. In this way, the LIGphotoelectrode could be used for sensitive photoelectrochemical bio-sensing, demonstrating a simple approach for free-standing photo-electrodes of various applications.
4. Laser induced graphene for multimodal sensor
The general biosensors that are specifically designed for a certainphysical/chemical parameter, can be totally irresponsive to other pa-rameters [103]. In order to provides amore comprehensive perspective,signals from chemical sensors and physical sensor are normally incor-porated [104]. However, the incorporation process usually involvesmultiple complicated integration of diverse materials and layers,which may prevent mass-production and deteriorate the sensing accu-racy of the devices [105]. Compared to the biosensor that are specificallydesigned for one variable while totally irresponsive to other variables,integrated biosensor assays had been reported tomeasuremultiple var-iables at one time [103,106]. The LIG technology by being based on onemain precursor and the laser direct writing process, variousmultimodalsensors can be fabricated in a straightforwardmanner, avoiding the po-tential deterioration on device performance and complicated integra-tion optimization.
Recently, wearable sweat sensors had drawn much attentions[107,108], due to its rapid, continuous and non-invasive health moni-toring for a wealth of physiologically relevant information [109]. Gaoet al. reported a wearable hybrid sweat sensor, consisting of tempera-ture sensor, strain sensor, and uric acid (UA) and tyrosine (Tyr) sensorthat are all based on LIG, Fig. 8(a-h) [105]. For temperature sensing,the conductivity of LIG was enhanced with the increased temperatureowing to increased electron–phonon scattering and thermal velocityof electrons in the sandwiched layers [110]. For strain sensing, the po-rous structure under an external strain will deform and enhance con-tacts, resulting in a decreased resistance. The rapid and accuratedetection of UA and Tyr in human sweat in situ, which are relatedwith diabetes, gout, metabolic disorders and other diseases wasachieved due to fast electron mobility, high current density and ultralarge surface area of LIG [105]. To continuously measure temperature,respiration rate and low concentrations of UA and Tyr, a microfluidicplatform was engraved on PI film with the laser for dynamic sweat
10 Z. Wan et al. / Sustainable Materials and Technologies 25 (2020) e00205
Fig. 8. (a)Multiple functions of the sensor: ultrasensitive sweat UA and Tyr detection, sweat rate estimation, temperature sensing and vital-sign (for example, heart rate and respirationrate) monitoring. (b) Photographic image of a flexible lab-on-skin patch. Scale bar, 1 cm. (c-d) SEM images of the LIG for chemical sensing. Scale bars, 200 μm (c) and 3 μm (d). (e-f)Mechanisms of temperature sensing (e) and strain sensing (f) using LIG. (g-h) UA (g) and Tyr (h) detection with the LIG in a 0.01 M ABS solution. Insets are the correspondingcalibration plots. Curr., current. Reproduced with permission [105]. Copyright 2019, Springer Nature. (i) The schematic drawing of multilayered working electrodes. Reproduced withpermission [106]. Copyright 2018, Elsevier. (j) Schematic of the fabrication of surface sensing process, including the fabrication of LIG electrodes, the Au coating, PANI coating and rGOcoating. Reproduced with permission [103]. Copyright 2018, American Chemical Society.
11Z. Wan et al. / Sustainable Materials and Technologies 25 (2020) e00205
sampling [105]. This entirely laser engraved sensor, which facilitatescalable manufacture and flexibility for the wearer's comfort, was vali-dated in physically trained and untrained subjects and demonstratedits potential for monitoring gout.
Park et al. demonstrated stretchable and flexible device with modi-fied LIG electrodes formonitoring the glucose level in sweat, pH sensing,and electrocardiogram (ECG), Fig. 8(i) [106]. The silver nanowire(AgNW) film were prepared between the PDMS and LIG to enhancethe electrical conductivity. The platinum and gold nanoparticles(PtAuNP) were deposited on the 3D porous LIG to improve the electro-chemical performance by enhancing the electron transfer rate and cata-lytic activity [111,112]. The various functional layers were deposited onthese LIG electrodes for sensing. For glucose sensing, the glucose oxi-dase enzymewas drop-cast onto the surface, exhibiting a low detectionlimit (5 μM) and a high linearity (0.99). For pH sensor, the PANI filmwere electrodeposited as a pH sensing membrane, showing a linear re-sponse (66 mV/pH) in the range from 4 to 7. For ECG sensor, the LIGelectrodes were utilized to detect the small electric current generatedby the contracting muscle for ECG signals, which provide useful
Fig. 7. (a) Schematic representation of the workflow employed on theMIP fabrication for the eformation of poly (EDOT), followed by amination, electropolymerization of EBT, in the presencSpectroscopy (EIS) measurements of MIP devices (left) and the corresponding calibration cuthe photoelectrochemical biosensing on TiO2-decorated LIG on the surface of an ITO(b) acetylcholinesterase (AChE), (c) acetylthiocholine (ATCl), (d) AChE + ATCl, (e) 0.1 ng mCP-pretreated AChE + ATCl. Reproduced with permission [99]. Copyright 2019, Royal Society
information about cardiovascular conditions [106]. The integrated bio-sensors demonstrate the potential of this novel fabrication techniqueand stretchable LIG metal nanocomposite for wearableelectrochemical-physiological hybrid biosensors [106].
Beside sweat sensor, Hu et al. reported amultiflavor sensing system,which was fabricated on PI using a laser direct witting and functional-ized with gold nanoparticles, reduced-graphene oxide, and polyanilinefor sensing NaCl, sugar, and vinegar, Fig. 8(j) [103]. In this sensor, theporous LIG as the electrode provides a large surface to volume ratioand achieves a high sensitivity. Gold nanoparticles were deposited toimprove the conductivity of the sensor and enhance its sensitivity[113]. Reduced graphene oxide composing a high density of edge-plane-like defect sites was dropped onto the electrode's surface andserved as the positive site in electron transfer to chemical and biologicalspecies [114], responding differently to Na+, Cl−, H+, CH3COO− ionsand sugar molecular. PANI, a conductive polymer, was employed tomodify the surface of LIG to detect vinegar solution, NaCl and sugarfor its ion selectivity [115–117]. By applying principal component anal-ysis, this sensing system shows a high sensitivity and selectivity with
lectrochemical biosensor for detection of CAP: electrodeposition of EDOT with subsequente of CAP or without CAP, and finally template removal. (b-c) Electrochemical Impedancerves (right). Reproduced with permission [98]. Copyright 2019, Elsevier. (d) Principle of. (e) Photocurrents of the LIG biosensor in (a) blank PBS and PBS containing
L−1 chlorpyrifos (CP)-pretreated AChE + ATCl, (f) 0.1 ng mL−1 CP, and (g) 1.0 ng mL−1
of Chemistry.
12 Z. Wan et al. / Sustainable Materials and Technologies 25 (2020) e00205
the detection limit of 0.1 mM for NaCl and sugar solutions, and 10−4
times diluted commercial table vinegar solution (5% acetic acid) [103].
5. Conclusions and outlook
In this paper, we first discussed the fabrication of LIG, the mecha-nism and the strategies to engineer the properties of LIG by the regula-tion of laser parameters, atmosphere, doping and surface modification.Due to the 3D porous structure, the good conductivity of LIG, and thesimple fabrication with laser patterning process in laboratory and in-dustrial scale, various state-of-the-art LIG biosensors have been re-ported in the literature and summarized in this review. The LIGelectrodes were employed directly or were modified with ion-selective membranes, catalytic nanoparticles, aptamer or enzyme forsensing (AA, DA, UA, H2O2, Urea, BA, ions, PBA, BPA, CAP and AChE in-hibitor). In the end, the integrated LIG biosensors combining severalsensing functions (temperature, strain, glucose, microfluids, pH, ECG,muti-flavors) were also demonstrated. Considering that the researchin this field is still in infancy and is advancing with a rapid pace, thereis plenty of room for further development of LIG biosensors for detect-ing biomarkers such as DNAs, RNAs, proteins, peptides as well aswhole cells. Also, the performance of LIG such as its electrocatalytic ac-tivity can be optimized by controlling the porosity, composition, mor-phology or surface modification. Currently, most LIGs are fabricated bylaser ablation directly under ambient air condition. When the powerdensity of incident laser exceeds the threshold ionization of dielectricmedia, the plasma can be generated on the focal surface, resulting in asubsequent combination of thermal andmechanical shock on themate-rials [118]. For high-power laser induced plasma, the plasma can be in-duced by the laser and subsequently focuses on the sample surface,producing the LIG immersed in a dielectric medium such as water[119]. To date, most current measurements rely on electrochemicaltest. Other methods of analysis such as fluorescence or field-effecttransistor-based biosensor should also be explored for LIG biosensing.We expectmuchmoremultifunctional LIG biosensorswill be developedin the near future because of the beauty of this simple fabrication pro-cess of laser directwriting and thematuring functionalization strategiesfor multiplexing. Finally, a LIG biosensor could be integrated with otherflexible LIG devices such as supercapacitor for potential one-stepmanufactured portable electronics for applications in safety, healthcare and environment monitoring.
Declaration of Competing Interest
The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.
Acknowledgements
The authors acknowledge the funding support of Australian ResearchCouncil Industry Transformational Research Hub (IH 180100002).
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