(PDF) Exploring Graphene and MoS 2 Chips Based Surface Plasmon Resonance Biosensors for Diagnostic Applications

REVIEW published: 26 August 2020 doi: 10.3389/fchem.2020.00728 Exploring Graphene and MoS2 Chips Based Surface Plasmon Resonance Biosensors for Diagnostic Applications Devi Taufiq Nurrohman 1,2 , Ying-Hao Wang 1 and Nan-Fu Chiu 1,3* 1 Laboratory of Nano-photonics and Biosensors, Institute of Electro-Optical Engineering, National Taiwan Normal University, Taipei, Taiwan, 2 Department of Electronics Engineering, State Polytechnic of Cilacap, Cilacap, Indonesia, 3 Department of Life Science, National Taiwan Normal University, Taipei, Taiwan Until now, two-dimensional (2D) nanomaterials have been widely studied and applied in the biosensor field. Some of the advantages offered by these 2D materials include large specific surface area, high conductivity, and easy surface modification. This review discusses the use of 2D material in surface plasmon resonance (SPR) biosensor for diagnostic applications. Two-dimensional material reviewed includes graphene and molybdenum disulfide (MoS2 ). The discussion begins with a brief introduction to the Edited by: Yu Jiang, general principles of the SPR biosensor. The discussion continues by explaining the Harvard Medical School, properties and characteristics of each material and its effect on the performance of United States the SPR biosensor, in particular its sensitivity. This review concludes with some recent Reviewed by: applications of graphene- and MoS2 -based SPR biosensor in diagnostic applications. Shuwen Zeng, Centre National de la Recherche Keywords: surface plasmon resonance, biosensor, 2D materials, graphene, diagnostic, MoS2 Scientifique (CNRS), France Jitendra Bahadur Maurya, National Institute of Technology Patna, India INTRODUCTION Kai Tong, Yanshan University, China The main challenge for all electrical, mechanical, and optical sensors is to detect chemical and biological analytes with low molecular weight (<400 Da) in very dilute conditions (Guo and Tan, *Correspondence: Nan-Fu Chiu 2009). Since surface plasmon resonance (SPR) biosensor was first introduced in the early 1990s,

it has proven to be one of the most powerful technologies for determining specificity, affinity, and kinetic parameters during binding of macromolecules in many types of bonds, including Specialty section: protein–protein (Kim et al., 2006), protein–DNA (Majka and Speck, 2006), enzyme–substrate or This article was submitted to inhibitor (Fong et al., 2002), receptor drug (Rich et al., 2002), lipid membrane–protein (Erb et al., Nanoscience, 2000), protein–polysaccharide (Beccati et al., 2005), and cell– or virus–protein (Zhang et al., 2014), a section of the journal among others. One of the advantages offered by this device is its unique ability to monitor molecular Frontiers in Chemistry binding activity in real time (Zeng et al., 2014). Received: 28 May 2020 The SPR biosensor is a type of biosensor that is very sensitive to changes in the refractive index Accepted: 14 July 2020 on the SPR sensing surface. The working principle of the SPR biosensor is based on the collective Published: 26 August 2020 coherent oscillation of free electrons in the metal conduction band first excited by the interactive Citation: electromagnetic field at the metal/dielectric interface, and the created charge density oscillation is Nurrohman DT, Wang Y-H and called surface plasmon polaritons (SPPs) (Raether, 1988). The SPPs will then form an electric field Chiu N-F (2020) Exploring Graphene that exponentially decays into the surrounding media with a depth of penetration in the range of and MoS2 Chips Based Surface Plasmon Resonance Biosensors for hundreds of nanometers. As a result, this evanescing electric field is very sensitive to changes in the Diagnostic Applications. surrounding refractive index. Thus, when there is a change in the refractive index of the medium, Front. Chem. 8:728. the characteristics (e.g., angle, wavelength, phase, etc.) of the light beam for SPR excitation will also doi: 10.3389/fchem.2020.00728 change (Zeng et al., 2014). Frontiers in Chemistry | www.frontiersin.org 1 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications There are several metals that can be used to excite SPPs metals (Cu, Ag, etc.) for a long time (about 1 year) in the including gold (Au), silver (Ag), copper (Cu), aluminum air and water environment (Kravets et al., 2014) because of its (Al), sodium (Na), and indium (In) (Raether, 1988; Maurya nature, which is impenetrable to most atoms and ions (Geim and Prajapati, 2016). Na is reactive in nature, In is very and Novoselov, 2007). This is very important for the purpose expensive, whereas Ag, Cu, and Al are susceptible to oxidation; of maintaining the quality factor during the functionalization in contrast, Au is resistant to oxidation and corrosion in process and biomolecular detection (Wu et al., 2019). different environments. Therefore, Au is the best choice as In this review, the authors summarize the current an active metal in conventional SPR sensor. However, bare development of 2D nanomaterials, namely, graphene and Au surfaces are not suitable for the biosensor because of its MoS2 in SPR biosensor. The discussion begins by discussing poor absorbance properties of biomolecules (Wu et al., 2010). the general principles of the SPR biosensor. The discussion Therefore, traditional biosensors are not capable to detect low continues by explaining the properties and characteristics of each molecular weight of biomolecules because of poor attachment material and its effect on the performance of the SPR biosensor, of these biomolecules to the bare metal surface (Maurya and in particular its sensitivity. This review concludes with some Prajapati, 2016). Until now, many methods have been developed recent applications of graphene- and MoS2 -based SPR biosensor to increase the sensitivity of SPR sensor such as using adhesion in diagnostic applications. layer (Agarwal et al., 2016a,b), metal nanoparticles and nanohole (Prasad et al., 2019), metal nanoslits (Yeung et al., 2018), and GENERAL PRINCIPLE OF SPR BIOSENSOR gold nanoparticles (Amendola et al., 2017). But until now, precise control over the geometry and optical properties of these There are several types of SPR biosensor platforms. Some of nanostructures is still very challenging (Kasani et al., 2019). them are attenuation-total reflection, optical prism couplings, Recently, there have been many publications on SPR optical fiber couplings, grating couplings, and others. Of the biosensors that use thin films with high refractive index [Si many platforms, the prism coupling based on Kretschmann and two-dimensional (2D) materials such as graphene and configuration has become standard technique to excite SPPs molybdenum disulfide (MoS2 )] to increase sensor sensitivity (Prabowo et al., 2018). In this configuration, the metal is usually (Tabasi and Falamaki, 2018). Two-dimensional materials such as deposited on the surface of the prism. After that, the prism is graphene and MoS2 have unique properties and offer promising illuminated by light which is p-polarized with a certain angle of opportunities. Graphene has a high surface-to-volume ratio, incidence (Figure 1A) (Damborský et al., 2016). By changing the which will produce strong interactions with biomolecules, angle of incidence, a sharp decrease is found in the intensity of excellent transparency, electron conductivity, and superior reflected light for certain range of incident angles. The angle at mobility (>2 × 105 cm2 V−1 s−1 at electron density 2 × which the minimum reflected light is called the SPR angle, which 1011 cm−2 ), large specific surface area (>2,500 m2 g−1 ), large in theory can be determined by the equation: Young’s modulus (>0.5–1 TPa), and high thermal conductivity (>3,000 W mK−1 ) (Wang et al., 2019). Besides graphene, MoS2 s ! is another 2D material that has recently been used for SPR −1 1 n22 n2m θSPR = sin applications. This material has higher optical absorption than n1 n22 + n2m graphene with exceptional optical and electrical properties (Ouyang et al., 2016). More importantly, the cytotoxicity and where θSPR shows the SPR angle; n1 and nm , respectively, genotoxicity of MoS2 are considered relatively low to most indicate the refractive indices of prism and metal. Adsorption biospecies, which is the pre-requisite for the applications in and desorption occurring on metal surfaces change the refractive biosensing (Kaur et al., 2018; Hu et al., 2019). index of the near media metal–dielectric interface and change Many researchers report that individual graphene and MoS2 the SPR angle (Figure 1B). Therefore, monitoring of changes can increase the sensitivity of SPR through a simulation in the SPR angle can be used to analyze adsorption–desorption approach. Verma et al. (2015) reported that by adding activities or associations that occur on metal surfaces (Tang and graphene to the gold surface, the sensitivity of the sensor Zeng, 2010). had increased from 30.85 to 33.98◦ /(RIU: refractive index Monitoring of adsorption and desorption activities on the unit). Similar results were also reported by Maurya and metal surface of the SPR is expressed in a curve called a Prajapati (2016). They compared the sensitivity of SPR on four sensogram as shown in Figure 1C. Sensogram can be obtained different structures, namely, bare Ag, Ag/graphene, Ag/MoS2 , based on changes in SPR angle or changes in reflectivity at and Ag/MoS2 /graphene. At the change of the refractive index of any time due to biomolecular interactions (Schasfoort, 2008). 0.068, the SPR angle shifts in the four structures were 4.38, 4.41, The monitoring process using this sensogram begins with 4.56, and 4.61◦ . Based on these results, MoS2 individuals showed the sensor surface conditioning using an appropriate buffer better sensitivity than graphene. But the best performance is in solution to create a baseline and activate a ligand that functions the structure composed of MoS2 and graphene (Maurya and as a bioreceptor to capture the target analytes. The next Prajapati, 2016). Similar results were also shown by other metals step is to inject the analyte onto the sensing surface. The such as Cu and Au (Maurya et al., 2015; Zeng et al., 2015; target molecule will be selectively captured by the ligand. The Maurya and Prajapati, 2016). In addition, some researchers also more molecules captured, the higher the SPR angle/reflectivity claim that 2D materials such as graphene can protect reactive changes. Next, a buffer is injected into the sensor and the Frontiers in Chemistry | www.frontiersin.org 2 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications FIGURE 1 | (A) Instrument setup for an SPR experiment. (B) Change in the SPR angle of incident light from angle a to angle b on the binding of an analyte molecule to a bioreceptor molecule. (C) Response of the SPR experiment in the form of a sensogram. Figures (A–C) were reproduced from Patching (2014) with permission from Elsevier. Copyright 2014, Elsevier. unattached component specifically flows during the dissociation flexibility, and it also has weak out-of-plane π bonds responsible phase. In this step, also the dissociation phase of the analyte for its thermal carrying, electrical charge, and transparency begins. Finally, a regeneration solution is injected to break (Amieva et al., 2016). When compared with conventional noble the specific bond between the analyte and the ligand. If metals such as Au, Ag, Cu, Cr, and Al, graphene has low energy ligands are properly immobilized on the sensor surface, they losses (e.g., Ohmic loss and radiative loss) and good tunability. remain on the sensor, whereas the target analytes are removed The confinement of the surface plasmons (SPs) in the graphene quantitatively (Ritzefeld and Sewald, 2012; Patching, 2014). Based is much stronger than in conventional noble metals (Luo et al., on this sensogram curve, several parameters can be obtained 2013). All of these advantages make graphene a promising including the association rate constant (K on ), the dissociation material for future sensor applications. Recently, graphene has rate constant (K off ), and the equilibrium dissociation constant emerged as an alternate plasmonic material but only in the (K d ) (Moscetti et al., 2017). terahertz to mid-infrared range (Gupta et al., 2019). In SPR biosensor, a plasmonic metal that is functionalized GRAPHENE-BASED SPR BIOSENSOR with graphene has four advantages, namely, (i) graphene has a very high surface-to-volume ratio, which is expected to be Graphene Properties and Their Potential in beneficial for efficient adsorption of biomolecules compared the SPR Biosensor with bare metal; (ii) graphene increases the adsorption of Graphene is the name of a single layer of carbon atoms organic and biological molecules because their carbon-based ring arranged in a 2D crystalline hexagonal lattice due to the sp2 structure enables π stacking interaction with the hexagonal cells hybridization of carbon. Thus, graphene has strong in-plane of graphene; (iii) controlling the number of graphene layers σ bonds, responsible for its high mechanical strength and transferred on to the plasmonic metal interface enables control Frontiers in Chemistry | www.frontiersin.org 3 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications of the SPR response and the sensitivity of SPR measurements 2014; Maurya and Prajapati, 2020). For example, in the case of (Szunerits et al., 2013); (iv) the presence of graphene on top of prostate cancer, the prostate-specific antigen has a concentration plasmonic metal can be used to protect metals from oxidation of 4 ng/mL in the blood. The development of biosensors is so that the stability and quality factor of plasmonic metal can be currently leading to efforts to achieve sensors that are as sensitive maintained (Szunerits et al., 2013; Kravets et al., 2014; Wu et al., as possible (Metkar and Girigoswami, 2019). 2019). In the SPR biosensor, the electromagnetic field is an Graphene has high electron transport mobility and a high evanescent wave that decays exponentially into both the metal surface-to-volume ratio. Electrons move at a speed of 1 million and sensing layer regions. An evanescent electromagnetic field meters per second (highest mobility ∼ 200,000 cm2 /Vs). This enhancement leads to the increase in the SPR sensor sensitivity property makes it possible to be a future sensor with ultrafast to perturbations in the sensing layer refractive index. In addition, speed. Because this is a 2D material, each graphene atom can be the increase in the sensing layer refractive index causes the considered a surface atom, and as a result, each atomic site can increase in the SPR sensor sensitivity (Tabasi and Falamaki, be involved in biomolecular interactions. This graphene feature 2018). Shalabney and Abdulhalim (2010) investigated the effect can ultimately be responsible for the response of ultrasensitive of material with a high refractive index (silicon) with a thickness sensors with the lowest detection capability to even one single of 10.5 nm, which was deposited on a metal surface. The results molecule (Basu and Bhattacharyya, 2012; Guy and Walker, 2016). obtained indicate that the presence of silicon increases the The use of pristine graphene has proved challenging because of intensity of evanescent electromagnetic fields and SPR sensitivity. difficult bottom-up synthesis (Smith et al., 2019), poor solubility The sensitivity of SPR produced on structures with and without (Yan, 2018), and agglomeration in solution due to van der Waals silicon are 200 and 67.5◦ /RIU, respectively (Shalabney and interactions (Skoda et al., 2014). As an alternative, compounds similar in structure to graphene can be synthesized from graphite or other carbon sources by a top-down method in an effort to achieve many of the advantages of pristine graphene while also imbuing the surface with functionalized oxygen groups. The oxidation of graphite in protonated solvents leads to graphite oxide, which consists of multiple stacked layers of graphene oxide (GO) (Smith et al., 2019). Graphene oxide has a hexagonal carbon structure similar to graphene but also contains hydroxyl (–OH), alkoxy (C–O–C), carbonyl (C–O), carboxylic acid (–COOH), and other oxygen- based functional groups. This oxygenated group is responsible for many advantages over graphene, including higher solubility and the possibility of easier surface functionalization with various types of bioreceptors. Several studies have reported that GO is compatible with single-strain DNA (ssDNA), peptides, and amino acids (Sharma et al., 2016). Through thermal, chemical, and electrochemical treatments, the oxygen functional groups in GO can be reduced to produce reduced GO (rGO). In rGO, the number of oxygen function groups is less than GO. Reduced GO can be considered as an intermediate structure between a pristine graphene and a highly oxidized GO, thus retaining some and losing some of the other properties of the two materials (Reina et al., 2017; Banerjee, 2018). Their interlayer distance was reduced from 7.9 Å on GO to 3.4 Å on rGO (Kitayama et al., 2019). By controlling the ratio of carbon to oxygen and the chemical composition in rGO, this material can be alternative for biological and biosensor applications. This can be done by selecting the reduction method in accordance with the expected properties (Pei and Cheng, 2012; Banerjee, 2018). Effect of Graphene on SPR Sensitivity Sensitivity can be defined based on the value of the limit of detection and the linearity of the biosensor. Limit of detection is very important because it shows the smallest concentration FIGURE 2 | Three fabricated chips. (A) Conventional chip. (B) GO-SPR chip. that can be detected by biosensors. In many cases today, (C) rGO-SPR chip. (D) SPR sensogram on conventional chip, GO-SPR chip, and rGO-SPR chip. Figures (A–D) were reproduced from Chiu et al. (2012) biosensors are required to be able to detect biomolecules in with permission from the SPIE. Copyright 2012, SPIE. the concentration range of ng/mL or fg/mL (Zagorodko et al., Frontiers in Chemistry | www.frontiersin.org 4 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications FIGURE 3 | Surface plasmon resonance sensograms obtained in response to BSA solutions of different concentrations flowing over the surfaces of the sensors. (A) Interaction with the conventional Au film-based (Au–MOA) sensor (1 mM). (B) Interaction with the 0.275 mg/mL GOS. (C) Interaction with the 1 mg/mL GOS sensor. (D) Interaction with the 2 mg/mL GOS sensor. Figures (A–D) were reproduced from Chiu and Huang (2014) with permission from Elsevier. Copyright 2014, Elsevier. Abdulhalim, 2010). Maharana et al. (2015) compared the increase injected into each chip. Based on the spectrum produced, the Au- in evanescent electromagnetic fields in silicon (10 nm) and Cys-GO chip shows the highest SPR angle shift. Surface plasmon monolayer graphene (0.34 nm) deposited in silver (43 nm). resonance angle shifts on GO-SPR, rGO-SPR, and conventional The fields produced on the surfaces of silicon and graphene SPR chips are 15,646, 2,312, and 5,418 mDeg, respectively. After monolayer are 375 and 825 (A/m)2 , respectively. In addition, NaOH is injected, the baseline on the GO-SPR chip is not at a wavelength of 653 nm, the SPR sensitivity on graphene is reduced. This shows a very strong covalent bond between the 340% higher than silicon. Based on the above data, it can be surface of Au-Cys-GO and TB DNA (Chiu et al., 2012). concluded that graphene in the SPR biosensor has a significant Based on the results of the above study, Chiu and role in increasing the evanescence electromagnetic fields and SPR Huang (2014) further performed GO variations to detect sensitivity (Maharana et al., 2015). immobilization of bovine serum albumin (BSA). Graphene Research on graphene-based SPR biosensor in the group oxide with concentrations of 0.275, 1, and 2 mg/mL were led by Professor Chiu has been started since 2012. Preliminary immobilized on the gold surface using cystamine linker. research in this group is to investigate SPR biosensor on single- Furthermore, the carboxyl group on GO is activated using layer GO and rGO to detect tuberculosis bacterial DNA (TB 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and DNA). The study was carried out by investigating three different N-hydroxysuccinimide (NHS) with a concentration ratio of 4:1. chips, namely, GO-based SPR chip (GO-SPR), rGO-based SPR The results obtained indicate that all fabricated chips can detect chip (rGO-SPR), and conventional SPR chip. The cystamine BSA directly. At BSA concentrations of 100 pg/mL−100 µg/mL, dihydrochloride (Cys) ring was deposited on the gold surface the SPR sensogram shows that the higher the BSA concentration, to detect TB DNA on conventional chip and to immobilize GO the higher the SPR angle (Figure 3). The highest sensitivity found and rGO sheet on SPR chips (Figures 2A–C). Figure 2D is an on SPR chips with GO concentrations is 2 mg/mL. At this SPR sensogram that shows the SPR response after TB DNA is concentration, the SPR chip can detect BSA up to a concentration Frontiers in Chemistry | www.frontiersin.org 5 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications interactions. The immobilization procedure on the SPR chip and the results obtained are shown in Figures 5A–C. Based on Figure 5B, the SPR angle shift at the anti-BSA concentration of 1–100 µg/mL shows that the higher the anti-BSA concentration, the higher the SPR angle shift. Of the three chips fabricated, the Au/GO-COOH chip shows a higher response so that this chip has the best sensitivity. The initial conclusion of this experiment is that the presence of carboxyl groups on GO surfaces greatly influences the performance of the SPR biosensor. Authors tried to reduce the anti-BSA concentration to a concentration of 0.01 pg/mL. Linear curves are obtained when the anti-BSA concentration is 0.01–100 pg/mL. The results of this study indicate that GO-SPR chips modified with the carboxyl group have the best performance when compared with previous studies (Chiu et al., 2017a). Current Application of Graphene-Based SPR Biosensor Prabowo et al. (2016) developed a graphene layer to investigate DNA hybridization of Mycobacterium tuberculosis using the SPR biosensor. The detection mechanism is shown in Figure 6. The graphene layer is deposited on the SPR chip using the simple drop casting method. Furthermore, an ssDNA binds covalently to gold nano urchin (GNu) and forms a sensing probe called ssDNA– GNu. The binding mechanism of the graphene and ssDNA layers is caused by the existence of the π-π stacking force. When hybridization occurs between complementary ssDNA (cssDNA) and ssDNA, the hybridization force is more dominant than the π-π stacking force. The presence of cssDNA will disrupt the ssDNA–GNu from the graphene layer. The detection limit achieved from this experiment was 28 fM (Prabowo et al., 2016). In 2017, Chiu et al. (2017b) combined GO sheets with specific peptide aptamer to detect human chorionic gonadotropin (hCG) FIGURE 4 | (A) SPR experimental scheme to study the interaction of BSA and proteins. The surface functionalization procedure for GO with anti-BSA. (B) Equilibrium analysis of binding of anti-BSA and BSA. Figures peptide is shown in Figure 7. In this study, authors used (N-) (A,B) were reproduced from Chiu et al. (2014) with permission from the PPLRINRHILTR (-C) (N-Pro-ProLeu-Arg-Ile-Asn-Arg-His-Ile- Nanoscale Research Letters. Copyright 2014, Springer Nature. Leu-Thr-Arg-C) to assay hCG protein. To block potential sites of interaction, the remaining carboxyl groups that are activated on of 100 pg/mL, which is 4.3 times greater when compared to the surface of the GO sheet are blocked by injecting ethanolamine conventional chips [Au–mercaptooctanoic acid (MOA)]. solution. From this experiment, the limit of detection obtained In the same year, Chiu et al. (2014) applied a GO-based was 0.065 nM with sensitivity 16 times higher than conventional SPR biosensor that had been developed previously to study SPR chips. the interaction of antibody and antigen. In this study, they In 2019, Chiu et al. (2019a) detected the same protein (hCG) studied the interaction between BSA and anti-BSA. The study using a modified GO-based SPR chip by adding a carboxyl group was conducted by comparing GO-SPR chip and conventional to the sensing surface (Figure 8). Based on the results of previous chip that had been developed previously. Based on Figure 4, the studies, the carboxyl group on the sensing surface produces high SPR angle shift at the anti-BSA concentration of 75.75 nM on affinity and stronger binding of biomolecules. To test biosensor the GO-SPR chip is 1.4 times higher than conventional chip. At selectivity, hCG protein was mixed with 20 nM BSA and 20 nM the highest concentration (378.78 nM), the change in SPR angle Human serum albumin (HSA). Based on the calibration curve, on GO-SPR chip is two times higher than conventional chip. This there is no significant interaction between peptides with BSA and shows that GO-SPR chip is more sensitive than conventional chip HSA. This shows high selectivity and a strong bond between so that this GO-SPR chip has the potential to be used in clinical peptides and hCG. The limit of detection for hCG in clinical diagnoses with lower concentrations. serum samples is 1.15 pg/mL. In 2016, Chiu et al. (2017a) modified the GO-SPR chip Chiu et al. (2018) also used carboxyl-GO–based SPR that was developed previously by adding the carboxyl group immunosensor to detect non–small cell lung carcinoma through (–COOH) to the SPR chip to study the anti-BSA and BSA cytolerayin 19 (CK19) protein biomarkers in spiked human Frontiers in Chemistry | www.frontiersin.org 6 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications FIGURE 5 | (A) Fabrication of SPR chip with biomolecular immobilization on modified surface of carboxyl-functionalized GO film. (B) Analysis of antigen-antibody interaction on various sensing chips with various analyte concentrations. (C) Sensogram and calibration curve on Au/GO-COOH chip at concentrations of 0.01–100 pg/mL. Figures (A–C) were reproduced from Chiu et al. (2017a) with permission from Elsevier. Copyright 2017, Springer Nature. plasma. In this immunosensor, small amounts of CK19 specific These results reaffirm that the presence of carboxy groups on the antibodies are immobilized on the SPR chip to specifically detect GO surface has been shown to increase biosensor sensitivity. CK19 protein (Figure 9). Next, CK19 protein with different In 2019, Chiu et al. (2019b) developed a carboxyl-GO– concentrations is injected into a functioning SPR chip. Based on based SPR immunosensor to detect pregnancy protein-associated the SPR angle response at each CK19 protein concentration, it plasma protein A2 (PAPP-A2) in human blood plasma. The was concluded that the SPR immunosensor had good linearity carboxyl-GO surface was functionalized by utilizing covalent at a CK19 concentration of 0.001–100 pg/mL (Chiu et al., 2018). bonds between carboxylic acid and anti–PAPP-A2 protein. In Frontiers in Chemistry | www.frontiersin.org 7 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications FIGURE 6 | The cssDNA detection mechanism developed by Prabowo et al. This figure was reproduced from Prabowo et al. (2016) with permission from Elsevier. Copyright 2016, Elsevier. FIGURE 7 | Surface modification to detect hCG protein with SPR biosensor. Figures (A–D) were reproduced from Chiu et al. (2017b) with permission from Elsevier. Copyright 2017, Elsevier. addition, BSA was covalently immobilized to block carboxyl-GO highest SPR angle shift lies in the PAPP-A2 protein. The SPR sheets in areas that are not coated with anti–PAPP-A2 protein. angle shift in other types of proteins is much smaller. Therefore, Figure 10A shows how the sensor selectivity is generated. Of the it can be concluded that the SPR immunosensor developed six types of proteins that are injected on the sensing surface, the has good selectivity. After that, the accuracy and precision of Frontiers in Chemistry | www.frontiersin.org 8 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications FIGURE 8 | Schematic illustration of the conversion of (A) GO into (B) carboxyl-GO sheets via a facile one-step chloroacetic acid modification route. (C) GO surface activation with EDC/NHS. (D) The attachment of peptides via amine coupling and the deactivation of the unreacted surface sites. (E) Immobilization of the peptide on the carboxyl-GO–based SPR chip using non-immunological to detect hCG protein. (F) Schematic instrumental setup of the Kretschmann configuration. Figures (A–F) were reproduced from Chiu et al. (2019a) with permission from Dove Medical Press. second linear range with high concentration has a regression equation y = 8.34x + 4.42 with a correlation coefficient of 0.991, where y represents the SPR angle and x represents the PAPPA2 concentration. Based on the calibration curve, this developed chip can used to quantitatively analyze the concentration of PAPP-A2 protein in spiked human plasma up to concentration of 0.01 pg/mL. In 2020, Fan et al. (2020) detected PAPP-A and PAPP-A2 using GO-based SPR biosensor. To be able to detect PAPP-A and PAPP-A2, anti–PAPP-A and anti–PAPP-A2 were immobilized on the GO surface. Tests carried out in this experiment include the sensitivity and selectivity of biosensor in protein mixtures. Figure 11 shows the SPR response curves on traditional chip and GO-SPR chip for PAPP-A, PAPP-A2, and protein mixture sample (CK-19, HSA, hCG, CA 19-9, PAPP-A, PAPP-A2). Based on Figure 11A, when detecting PAPP-A, the SPR angle on the GO- SPR chip shows a better response than traditional chip. The same FIGURE 9 | Graphene oxide–COOH sheet-based SPR immunosensor to response also occurs when detecting PAPP-A2 (Figure 11B). detect CK19 protein. The figure was reproduced from Chiu et al. (2018) with This shows that the GO-SPR chip has better sensitivity than permission from Elsevier. Copyright 2018, Elsevier. traditional chip. Furthermore, in the protein mixture sample, the SPR biosensor response to the detection of PAPP-A and PAPP-A2 showed a curve that was almost the same as the sample that was not mixed. Based on these data, it is proven that, in addition to the GO-carboxyl–based SPR chip are tested based on the SPR sensitivity, biosensor selectivity also has good performance. The angle response at different concentrations of PAPP-A2 protein limit of detection in GO-based SPR biosensor in this experiment (Figure 10B). The results show that there are two ranges where is 0.5 ng/mL. the calibration curve produces good linearity. The first linear The description above shows that, to be able to range with low concentration has a regression equation y = selectively detect a certain biomolecule, an appropriate 2.25x + 5.78 with a correlation coefficient of 0.989, whereas the bioreceptor is needed. Table 1 below shows a summary of Frontiers in Chemistry | www.frontiersin.org 9 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications FIGURE 10 | (A) The SPR angle shift plot of six different proteins to determine biosensor selectivity. (B) Calibration curve of the average SPR response to various PAPP-A2 protein concentrations. Figures (A,B) were reproduced from Chiu et al. (2019b) with permission from the Dove Medical Press. FIGURE 11 | Surface plasmon resonance response curves and curve fitting equations of PAPP-A (A) and PAPP-A2 (B) measurement with the traditional SPR biosensor and GO-SPR biosensor. Figures (A,B) were reproduced from Fan et al. (2020) with permission from Dove Medical Press. research results on graphene-based SPR biosensors and the fulfill. The second chip is a GO-based chip. Graphene oxide results obtained. can be immobilized on a metal surface by using a modified Based on Table 1 above, to date, there are many graphene- chemical covalent bond immobilization method. By using this based SPR chips that have been fabricated by researchers. In method, the chip and GO bind with a very strong force, our laboratory, chips that have been successfully fabricated are so it is not easy to fall off, and repeated detection is very graphene, GO, and GO-COOH–based SPR chips. On graphene- possible. However, the thickness of GO grown by this method based SPR chip, graphene is usually immobilized on a metal is very difficult to control. The last chip is GO-COOH–based surface using the chemical vapor deposition method and the chip. This chip can be fabricated using a modified chemical method transferred by electrostatic adsorption. The advantages covalent bond immobilization method. The SPR chip fabrication offered by this method include the ease of controlling the process is moderate but produces SPR chip with very high thickness of graphene. But the chip fabrication process is quite biocompatibility. In addition, this method can make the wafer complex. In addition, the force produced between graphene and and GO-COOH a super strong binding force and not easy chip is van der Walls force or electrostatic force. This force to fall off and can be used for repeated detection. But the is weak enough so that repeated detection becomes difficult to thickness of the GO-COOH layer is not easily controlled. In Frontiers in Chemistry | www.frontiersin.org 10 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications TABLE 1 | Graphene-based SPR biosensor design, detection limits, and the resulting linear range. SPR system Bioreceptor Target Limit of Linear range References detection Au/grapheme α-thrombin aptamer α-thrombin 0.05 nm — Wang et al., 2011 Au/graphene/gold nanostars ssDNA cssDNA 500 Am — Zagorodko et al., 2014 Au/grapheme Cholera toxin antigen Anticholera toxin 4 pg/mL 0.004–4 ng/mL Singh et al., 2015 Au/grapheme ssDNA cssDNA 28 fM — Prabowo et al., 2016 Au/grapheme Antifouling folic acid Folic acid protein (FPA) 5 fM 5–500 fM He et al., 2016 Au/GO-COOH BSA Anti-BSA 0.01 pg/mL 0.01–100 pg/mL Chiu et al., 2017a Au/GO Peptide hCG protein 0.065 nM — Chiu et al., 2017b Au/SAM/GO/3ABA Anti–gelactin-3 Gelactin-3 2 ng/mL — Primo et al., 2018 Au/GO-COOH Lung cancer antibody CK19 protein 0.001 pg/mL 0.001–100 pg/mL Chiu et al., 2018 Au/GO-COOH PAPP-A2 Anti–PAPP-A2 0.01 pg/mL 0.01–10.000 Chiu et al., 2019b pg/mL Au/GO Anti–PAPP-A PAPP-A 0.5 ng/mL — Fan et al., 2020 Anti–PAPP-A2 PAPP-A2 0.5 ng/mL — TABLE 2 | Graphene-based SPR chip, which has been successful in fabrication, advantages, and disadvantages. SPR system Chip process Repeatable detection Thickness and precision of References film making Au/grapheme Complex Difficult Easy to control Wang et al., 2011 Au/graphene/gold nanostars Complex Difficult Easy to control Zagorodko et al., 2014 Au/grapheme Complex Difficult Easy to control Singh et al., 2015 Au/grapheme Complex Difficult Easy to control Prabowo et al., 2016 Au/grapheme Complex Difficult Easy to control He et al., 2016 Au/GO-COOH Moderate Feasible Difficult Chiu et al., 2017a Au/GO Easy Feasible Difficult Chiu et al., 2017b Au/SAM/GO/3ABA Easy Feasible Difficult Primo et al., 2018 Au/GO-COOH Moderate Feasible Difficult Chiu et al., 2018 Au/GO-COOH Moderate Feasible Difficult Chiu et al., 2019b Au/GO Easy Feasible Difficult Fan et al., 2020 other research groups, there are other types of SPR chips energy dispersion, integrated circuits, photosensitivity, and that have been successfully fabricated. Our analysis regarding highly efficient emitter (Li and Zhu, 2015; Kalantar-Zadeh and the advantages and disadvantages of each chip is shown Ou, 2016). in Table 2. Lately, MoS2 has attracted the attention of researchers in the field of optical biosensors because of its high electron conductivity, tunable band gap, and high optical absorption MoS2 BASED SPR BIOSENSOR efficiency. As a monolayer of MoS2 possesses a higher optical absorption efficiency (∼5%) than that of graphene (2.3%) (Lopez- MoS2 Properties and Their Potential in the Sanchez et al., 2013), it can promote plasmon excitation through SPR Biosensor an efficient charge transfer between MoS2 and the thin metallic Two-dimensional MoS2 is an inorganic compound composed of film (Kim et al., 2019). In addition, the large surface area and the molybdenum (Mo) and sulfur (S) (Das et al., 2015). This material presence of free sulfur atoms are typical features of MoS2 , which is a semiconductor material with an ultrathin direct band make it a potential material for developing biosensing interfaces gap and belongs to the transition metal dichalcogenide group. (Kaushik et al., 2019a). When MoS2 layers are deposited on metal Molybdenum disulfide has characteristics similar to graphene; it thin films, the strong coupling can be induced at the metal/MoS2 is not affected by dilute acid or oxygen and is not reactive with interface because of the effective charge transfer and large electric other chemicals (Van Santen and Neurock, 2017). It also has the field enhancement, which will result in increased SPR sensitivity unique characteristics of electrical and photo-responsiveness of (Hu et al., 2019). In addition, the MoS2 layer serves to inhibit the Shockley-type surface state properties. Therefore, MoS2 has been penetration of oxygen and water molecules to prevent oxidation widely studied with regard to SP-enhanced photoluminescence, of aluminum and silver metals (Xu et al., 2018; Kim et al., 2019). Frontiers in Chemistry | www.frontiersin.org 11 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications FIGURE 12 | Surface plasmon resonance signals of (A) bare Ag and (B) Ag/MoS2 chip in water with laser irradiation. Figures (A,B) were reproduced from Kim et al. (2019) with permission from MDPI. FIGURE 13 | Surface plasmon resonance signal against BSA in phosphate-buffered saline (PBS) solution of the (A) optical fiber SPR biosensor without MoS2 and (C) developed SPR biosensor. Calibration curve of (B) Ab/gold/fiber; (D) Ab/MoS2 /gold/fiber against varying concentration of BSA in PBS solution. Figures (A–D) were reproduced from Kaushik et al. (2019a) with permission from the Springer Nature. Frontiers in Chemistry | www.frontiersin.org 12 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications Effect of MoS2 on SPR Sensitivity monochloroacetic acid (MCA)] to form a new sheet called MoS2 - In 2019, Kim et al. (2019) used a thin silver layer to excite SP COOH sheet. Briefly, the experiments carried out are illustrated waves. They compared two different chips, namely, conventional in Figure 14 below. The MoS2 -COOH sheet is then applied chip (Bare Ag) and silver chip deposited with MoS2 (Ag/MoS2 ). Figure 12 shows the shape of the SPR signal measured every 5 min for 20 min on conventional chip and Ag/MoS2 chip. On conventional chip, the SPR signal shows a drastic change. This is presumably because the light that hits the prism triggers the oxidation of silver. Unlike the case with Ag/MoS2 chip, the SPR signal shows a consistent shape. This shows that the stability of the SPR chip on this chip is more stable than conventional chip. To determine the sensitivity produced, immunoglobulin G with a concentration of 600 nM is injected into each chip. Surface plasmon resonance angle shifts on conventional chip and Ag/MoS2 chip are 0.20 and 0.25◦ , respectively. Based on this study, it can be concluded that the presence of MoS2 monolayer has been shown to increase SPR stability and sensitivity up to 125%. In the same year, Kaushik et al. (2019a) investigated the effect of MoS2 on the gold surface using optical fiber-based SPR biosensor to study interactions between anti-BSA and BSA. Experiments were carried out by injecting BSA with different concentrations (10–50 µg/mL) on conventional chip and MoS2 - based chip. The resulting SPR signal and calibration curve are shown in Figure 13. The slope on the MoS2 -based SPR chip (0.9234) has a greater value than conventional chip (0.6139). In contrast, the limit of detection value on the MoS2 -based SPR chip has a smaller value (0.29 µg/mL) when compared to conventional chips (0.45 µg/mL). These results confirm that the presence of MoS2 on the SPR chip is proven to increase sensor sensitivity (Kaushik et al., 2019a). Current Application of MoS2 Based SPR Biosensor Development of MoS2 for SPR biosensors in a research group led by Professor Chiu started in 2018. Chiu and Lin (2018) FIGURE 15 | (A) The sensogram that shows the SPR response at different developed MoS2 functionalized with the carboxyl group by CYFRA21-1 protein concentrations. (B) Sensor selectivity for different types of proteins. Figures (A,B) were reproduced from (Chiu and Yang, 2020) with utilizing the sulfur vacancy in MoS2 . This vacancy will then be permission from Frontiers. filled by Cl originating from Cl–COOH [–COOH modified with FIGURE 14 | Synthesis of MoS2 -COOH sheet with monocholoroacetic acid (MCA). Figure was reproduced from Chiu et al. (2018) with permission from Elsevier. Copyright 2018, Elsevier. Frontiers in Chemistry | www.frontiersin.org 13 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications TABLE 3 | Molybdenum disulfide-based SPR biosensor design, detection limits, and the resulting linear range. SPR System Bioreceptor Target Limit of Detection Linear range Ref. Au/MoS2 /AuNPs Micro RNA-141 Micro RNA-141 0.5 fM - Nie et al., 2017 Au/MoS2 -COOH Anti-BSA BSA 1.45 pM 14.5 – 725 nM Chiu and Lin, 2018 Ag/MoS2 - immunoglobulin G - - Kim et al., 2019 Au/MoS2 Anti-BSA BSA 0.29 µg/mL - Kaushik et al., 2019a Au/MoS2 E. coli monoclonal antibodies E. coli 94 CFU/mL - Kaushik et al., 2019b Au/MoS2 /AuNPs Goat-anti-mouse IgG Mouse IgG 0.06 µg/mL Zhao et al., 2020 Au/MoS2 -COOH Anti-CYFRA 21-1 CYFRA 21-1 0.05 pg/mL 0.05 pg/mL – 100 ng/mL Chiu and Yang, 2020 CFU: Colony forming units. TABLE 4 | Molybdenum disulfide–based SPR chip, which has been successful in fabrication, advantages, and disadvantages. SPR system Chip process Repeatable detection Thickness and precision of References film making Au/MoS2 /AuNPs Complex Feasible Difficult Nie et al., 2017 Ag/MoS2 Easy Difficult Difficult Kim et al., 2019 Au/MoS2 Easy Difficult Difficult Kaushik et al., 2019a Au/MoS2 Easy Difficult Difficult Kaushik et al., 2019b Au/MoS2 /AuNPs Complex Difficult Difficult Zhao et al., 2020 Au/MoS2 -COOH Complex Feasible Difficult Chiu and Lin, 2018 Au/MoS2 -COOH Complex Feasible Difficult Chiu and Yang, 2020 to the SPR biosensor to detect protein antibodies. The results be immobilized directly on a metal surface. Therefore, thiol- obtained indicate that the MoS2 functionalization with –COOH and amine-group self-assembled monolayers of cystamine (Cys) can strengthen the SPR biosensor response up to 3.1 times when function as a bridge for immobilization of MoS2 -COOH sheets compared to conventional SPR chip (Chiu and Lin, 2018). on metal surfaces. A complete analysis related to the SPR chip Chiu and Yang (2020) used a single-layer MoS2 -COOH fabrication process, the advantages, and disadvantages are shown for signal amplification to detect lung cancer associated in Table 4. with the cytokeratin 19 fragment biomarker (CYFRA21- 1) using an SPR-based biosensor. To detect CYFRA21-1, MoS2 /COOH-based SPR chip was functionalized using lung CONCLUSION AND FUTURE RESEARCH cancer antibodies (anti-CYFRA21-1, TROMA-III). Figure 15 shows how biosensor performance and selectivity are produced. Based on the above review, it can be concluded that Based on Figure 15A, the greater the concentration of CYFRA21- 2D nanomaterial (graphene and MoS2 ) has been proven 1 (0 pg/mL−100 ng/mL), the greater the SPR angle shift. The experimentally to increase the sensitivity of the SPR biosensor. largest SPR angle shift occurs with the highest concentration Several experiments conducted in our laboratory have shown that (100 ng/mL). Based on Figure 15B, biosensor selectivity also GO or MoS2 -based SPR biosensors that are functional with the shows good results. The SPR angle shift on the CYFRA21-1 carboxyl group have been shown to increase sensor sensitivity. In protein shows a much greater magnitude than other types of several different detection applications (anti-BSA, anti-PAPPA2, proteins (CA-199, hCG, PAPP-A, PAPP-A2, and HAS) (Chiu and CYFRA 21-1, and CK19), the limit of detection shows the Yang, 2020). same level that is at the pg/mL level. This result can be used Research on the use of MoS2 in the SPR biosensor is still not as a foundation for a wider diagnostic application. Although, widely done. At present, most studies carry out only computer experimentally, graphene-based SPR biosensors have been widely simulations. Table 3 below shows a summary of research results used for a variety of applications, the mass production of SPR on the MoS2 -based SPR biosensors in our research group and chips still needs further research, especially those related to other groups conducting the same experimental study. efficiency in the fabrication process of SPR chips. The shape, To date, MoS2 -based chips fabricated by our research group size, number of layers, electronic band gap structure, purity, and have been successfully applied to detect protein antibody and graphene defects that grew in the experiment are all uncertain. CYFRA 21-1. The two chips that are fabricated are MoS2 These properties can influence the conductivity of the SPR chip that are functionalized with COOH. COOH can bind chip, interaction with biomolecules, and fluorescence quenching strongly to MoS2 due to strong bonds due to the presence of causing the performance of graphene-based SPR biosensors to be sulfur vacancies in MoS2 . Molybdenum disulfide–COOH cannot different for each fabrication. For MoS2 -based SPR biosensor, so Frontiers in Chemistry | www.frontiersin.org 14 August 2020 | Volume 8 | Article 728 Nurrohman et al. SPR Biosensor for Diagnostic Applications far it has not been possible to place monolayer of MoS2 uniformly The manuscript was written through contributions of all on a larger surface area. authors. All authors have given approval to the final version A high sensitivity detection device is needed as a pre- of the manuscript. cautionary measure before the spread of disease in the human body. For this purpose, the combination of some 2D material FUNDING can be one of the topics that is widely studied in the next few years. For example, the combination of MoS2 and This study was supported by the Ministry of Science and graphene has been shown to increase SPR sensitivity through a Technology of the Republic of China (ROC), Taiwan, for simulation approach. But so far, there have been no publications financially supporting this research under Contract No. MOST containing experiments on this structure. Not only in 2D 105-2221-E-003-027, MOST 106-2221-E-003-020, MOST 107- graphene and MoS2 material, but also other types of 2D 2221-E-003-009, MOST 108-2221-E-003 -020 -MY3, and MOST material such as WSe2 , MoSe2 , WS2 , and black phosphorous can 109-2221-E-003-028-MY3. be alternatives. ACKNOWLEDGMENTS AUTHOR CONTRIBUTIONS The authors would like to thank the Mackay Hospital, DN wrote the manuscript and performed the overall editing of Taipei, Taiwan, this work was approved by the Institutional the manuscript. N-FC provided guidance for the manuscript Review Board (IRB) of Mackay Hospital for Human Clinical setups and manuscript structure. N-FC and Y-HW oversaw the Trials (Permit Numbers: 15MMHIS020, 15MMHIS115, and project and performed the overall editing of the manuscript. 17MMHIS185). REFERENCES Chiu, N. F., Kuo, C. T., and Chen, C. Y. (2019a). High-affinity carboxyl-graphene oxide-based SPR aptasensor for the detection of hCG protein in clinical serum Agarwal, S., Giri, P., Prajapati, Y. K., and Chakrabarti, P. (2016a). Effect of surface samples. Int. J. Nanomedicine 14, 4833–4847. doi: 10.2147/IJN.S208292 roughness on the performance of optical spr sensor for sucrose detection: Chiu, N. F., Kuo, C. T., Lin, T. L., Chang, C. C., and Chen, C. Y. 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Express 18, 15458–15463. distributed under the terms of the Creative Commons Attribution License (CC BY). doi: 10.1364/OE.18.014395 The use, distribution or reproduction in other forums is permitted, provided the Xu, Y., Wu, L., and Ang, L. K. (2018). MoS2 -based highly sensitive near- original author(s) and the copyright owner(s) are credited and that the original infrared surface plasmon resonance refractive index sensor. IEEE J. publication in this journal is cited, in accordance with accepted academic practice. Sel. Top. Quantum Electron. 2:2868795. doi: 10.1109/JSTQE.2018. No use, distribution or reproduction is permitted which does not comply with these 2868795 terms. Frontiers in Chemistry | www.frontiersin.org 17 August 2020 | Volume 8 | Article 728