The impact of different gold nanoparticles (G NPs) structures on the plasmonic enhancement for DNA detection is investigated on a few-layer graphene (FLG) surface plasmon resonance (S PR) sensor. Two distinct structures of gold nanourchin s (GNu) and gold nanorods (GNr) were used to bind the uniquely designed single -stranded probe D NA (ssDNA) of Mycobacterium tuberculosis complex (M TBC ) DNA. The two types of GNPs -ssDNA mixture were adsorbed onto the FLG - coated S PR sensor through the π-π stacking force between the ssDNA and the graphene layer. In the presence of the complementary single -stranded D NA (cssDNA), the hybridization process took place and gradually removed the probes from the graphene surface. From S PR sensor preparation, the annealing process of the Au layer of the S PR sensor effectively enhanced the FLG coverage leading to a higher load of the probe D NA onto the sensing interface. The F LG was shown effective in providing a larger surface area for biomolecular capture due to its roughness. Carried out in the DNA hybridization study with S PR sensor, GNu , with its rough and spikey structures , significantly reinforced the overall D NA hybridization signal than the GNr with smooth superficies , especially in capturing the probe D NA. The D NA hybridization detection assisted by GNu rea ched the femtomolar range limit of detection (LoD). An optical simulation validated the extreme plasmonic field enhancement at the tip of the GNu spicules. The overall integrated approach of graphene - based S PR sensor and GNu -assisted D NA detection provided the proof -of-concept for the possibility for Tuberculosis disease screening using a low -cost and portable system potentially applied in a remote or the third world countries. Keywords: DNA biosensor , Mycobacterium tuberculosis , few-layer graphene , surface plasmon resonance, gold nanourchin s, gold nanorods 3 1. Introduction The world -shattering Coronavirus disease (COVID -19) pandemic has been an important reminder for the urging needs on the rapid screening and diagnostic methods of respiratory and airbo rne pathogens infection [1–3]. Another major threat from th e respiratory disease class is Tuberculosis (TB) infection , which typically spreads in numbers of poor and developing countries [4]. The shot-up of the morbidity and mortality rate of TB was predicted to increase significantly as the consequence of the reallocation of resources for the COVID -19 pandemic worldwide [5,6] . The M. tuberculosis complex (MTBC ) bacterium is highly noticed as the pathogen ic species causing the tuberculosis disease . It has been declared to bring out deadly effects that have infected almost a quarter of the world population , dominantly in the low-middle income countries in Africa, Asia, South America , and Eastern Europe [3,4] . Unfortunately , the human -to-human airborne transmission of this bacterium makes it hard to be tested in a rapid d iagnostic or screening protocol . One of the well -known D NA sequence biomarkers of the M TBC is the fragment of IS6110, which is stable for decad es from the mutation genetic part, and extremely specific to the M TBC species. This fragment exists in several copies in different locations of the M TBC genomic profile. This versatility has made this sequence a favorable biomarker for the PCR and sequence -based screening diagnostics. The polymerase chain reaction (PCR) , as the golden standard for quantification of the pathogen through specific nucleic acid amplification , is still regarded indirect, laborious , and expensive. These drawbacks have been the main challenges for the middle -low income countries to perform the routine and extensiv e PCR tests in society [1,7] . Additionally, the typical bench -top PCR system is fa r from portability means to be applied for T B cases , which most likely occur in 4 remote and harsh sites. Biosensor technology has been long spotlighted to provide an alternative to the time -consuming standard diagnostic methods , and its growth has been mome ntously accelerated by the current pandemic situation toward the realization of the next-generation diagnostics method s. Among myriads of disease biomarkers, the preference of the nucleic acid biomarker from the antigen is highly advantageous because its base-pairing hybridization is highly specific and robust [8] and easy to be functionalized with nanoparticles and other surfaces [9]. On the other hand, the detection of serology biomarkers requires complicated purification -separation protocols due to the complex protein matrices in blood or body liquid [10,11] . In the application of D NA on a biosensor, the uniform surface coverage by probe DNA molecules is essential to achieve the overall inherent biorecognition signal after hybridization with the target . The i nterfacial engineering for biomolecular capture s, such as via cross -linking agent [12], electrostatic attraction [13], nanoparticles ( NPs)-assisted capture [14], and many other methods have been crucially developed to achieve the best sensing device performance . Gowtham et al. reported that the mechanism of nucleobases physical absorption onto graphene and carbon nanotubes. Adenine (A), Thymine (T), Cytosine (C), and Guanine (G) interaction between the graphene layer were dominated by the molecular polarizability , which played a vital role in an attractive dispersion force between these materials [15]. The orientation of the binding was reported almost parallel to the graphene layer with a separation distance around 3.5 Å, and it indicated the interaction characteristic for the π-π stacking mechanism [15,16] . The aptamer binding on graphene was also studied for the α-thrombin monitoring application . The aptamer probes were functionalized by the amine or sulfide group and adsorbed on the graphene surface by the π -π stacking force mechanism. Subsequently, the presence of the α-thrombins attracted the labeled - aptamer from the graphene surface due to a massive covalent bon d between the amino or sulfide 5 group to the α -throm bin[17]. In additi on, the nanomolar and sub -nanomolar range detection of DNA hybridization were reported as well by the use of graphene for surface -assisted adsorption of the D NA strand [18,19] . The utiliz ation of solution -based graphene. Such as graphene oxide (GO) for capturing the DNA strands via the π-π interaction was highly regarded as a simple and low -cost approach to initialize the biomolecular recognition on the sensor´s interface [20–22]. A significant challenge in working with solution -based graphene is transferring onto another solid substrate , such as the gold (Au) layer , typically used in the surface plasmon resonance (S PR) sensing setup . Song et al. recommended the improvement of the Au {111} crystallization on the surface to enhance the adsorption energy of the graphene on to the gold layer [23]. Another method emphasized the high possibility for colloidal graphen e onto the Au SPR. chip through metal -carbon bonding with a considerably high uniform coverage rate [20]. The application of deformed multi -layer graphene for effective biomolecular capture was also studied by Hwang et al., who successfully created electronic hotspots from the capture of D NA on the crumpled graphene layer [24]. Among the metallic nanoparticle clusters , AuNPs have gained significant attention to be combined with D NA in numbers of sensing platforms, such as in electrochemical, transisto r-based , and optical sensor s due to their high bio -affinity , chemical and physical stability , surface area to volume ratio , and excellent optical features [25–31]. Specifically , the plasmonic e nhancement from AuNPs has been applied in a variety of optical sensing setups, such as in S PR, photoluminescence , and surface -enhanced Raman spectroscopy (S ERS) biosensors showing ultrasen sitive D NA or aptamer detection [20,32 –35]. The plasmonic coupling is predominantly defined by nature, size, shape , structure, composition, and aspect ratio of the N Ps and the environment di electric constant and inter-spatial arrangement within the N Ps on a solid surface 6 [36]. Some studies reported the remarkable electric field enhancement of branched AuNPs with protruding spikes and sharp tips useful in biosensing configuration [37,38] . In this article, we presented the proof -of-concept of the M TBC screening method, using FLG on an SPR sensing system . The biorecognition signal from D NA was assisted by the tagging of probe D NA with gold nanoparticles (GNPs) with two distinct structures, gold nanourchins (GNu) or gold nanorods (GNr) , to be adsorbed onto the FLG layer via the π - π stacking. The hybridization event with the target D NA was c hecked through the release of the gold nanostructures -tagged probe from the sensing interface. The plasmonic activity from both gold nanostructures w as compared , and it was denoted that the GNu provided the most significant enhancement with an impressive l imit of detection (LoD). The proposed work combined both the ease of solution -based graphene integration onto the portable S PR chip and the facile detection methods for nucleic acid -based T uberculosis detection. Among experimental using clinical samples of the infectious diseases, that the future outlook will be performed in the high biosafety level laboratory, the screening and detection using biosensor technology can be a potential approach. Moreover, the modula r biosensing technology with the feature of components disposability can be an advantage to avoid contamination during the measurements . 2. Material and method The primary platform of the portable S PR biosensor utilizing organic light source in this experime nt was described in our previous reports and depicted in Fig. 1[39]. Modular based instrument s and its portability offer an advantage for the real detection s cheme of MTBC , which 7 commonly occurs in the remote area or third world countries. The disposability of the modular component is beneficial for the handling of the toxicity of the samples . 2.1. Material and Instruments Graphene powder and its dispersion solution were purchased from Angstron Materials Inc. (Ohio, U SA). The GNu with 70 nm size and GNr with the aspect ratio of 2.5 were bought from NanoSeedz (Hongkong , P.R.C ). DNA sequences were purchased from Purigo Biotech nology Co. (Taipei, Taiwan). The D NA probe and target sequence s were derived from the MTBC DNA fragment IS6110. The single -stranded ( ssDNA ) probe sequence used in this study was SH - CGTGCGGCTA TTACGAGGAC TCCACGCTGG (30 mers). The sequence of the complementary ssDNA (cssDNA) target was CCGAT AATGCTCCTG AGGTGCGACC (25 mers). In comparison, the sequence of mismatched single -stranded DNA (mssDNA) utilized in the specificity test was ACAGC ATTGCGCGTT CAGACACCGC (25 mers). The target D NA strand had been designed with shorter sequences (5 -mers shorter ) to accommodate the spacing activity in the probe DNA. The phosphate buffer saline (PBS) tablet from Sigma Aldrich (Missouri, U S) was employed to produce the PBS buffer solution. NaCl, K 2HPO 4, and KH 2PO 4 for salt buffer solution were purchased from Sigma Aldrich (Missouri, U S). The RAMaker system from Protrustech was applied to perform Raman spectroscopy.Co, Ltd (Tainan City, Taiwan) with charge -coupled device (C CD) camera system and coupled with Olympus micr oscope body. The collimation of light excitation was performed by 100 times objective lens (NA 0.5). The laser power source was 100 mW, the exposure time was 2 seconds , and the accumulation number is 3. The electron microscopy image was obtained from the f ield-emission scanning electron microscope (F ESEM ) with the instrument series of JSM-7500F (J EOL co., Tokyo, Japan), transmission electron 8 microscopes (TEM) with the instrument type of JEOL JEM -1230 and JEOL JEM -2100 PLUS (JEOL co., Tokyo, Japan). The Finite -difference time -domain (F DTD ) simulation was performed by Lumerical (Pensylvania, US) . Figure 1 is preferred in this location 2.2. Sensing chip preparation A gold sensing chip was prepared and produced based on the protocol explained in our previous report s [40]. Next, the chip was annealed in the baking chamber at 300 °C for 30 min with a very slow ramp -up of the temperature, followed by an immediate quenching process by exposing it to the N 2 gas at 20 °C. The quenching ramp -down temperature should be careful ly handl ed to avoid substrate crack. The annealing -quenching step are critical for improving Au {111} crystallization [23,41] . The d ispersant solution of graphene was diluted in the Deionized (DI) water to reach a 1% concentration. To prepare the F LG solution, t he graphene powder was then dispersed in 1% of the dispersant solution until reaching the concentration of 0.1 mg/mL before the ultrasonic treatment for 20 min s. Subsequently, the FLG solution was dropped on the sensing surface and baked at 80 °C (the boiling temperature point of the solution was avoided) until the dryness of the sensing layer was obtained . 2.3. The D NA immobilization and detection In this study, the ssDNA probe was tagged with the GNu before the adsorption onto the sensing membrane. First, a 70 nM GNu solution was centrifuged and suspended in the PBS solution with a rotation speed of 700 G for 30 minutes . The GNu solution was then mixed with the ssDNA in PBS until the final concentration of 100 nM ssDNA was reached. This mixed solution 9 was kept at room temperature for 20 -24 hours to allow the e ffective covalent binding between GNu and ssDNA probe. The target D NA was prepared through a serial dilution using the salt buffer to complete a series of concentration s from 100 fM to 100 nM. The salt buffer for target dilution was prepared by mixing 1 M K2HPO 4 and 1 M KH 2PO 4 in a 1 M NaCl solution . For GNr - assisted D NA detection, similar stages were applied by replacing the GNu with GNr solution. 3. Results and discussion 3.1. Few-layer graphene (F LG) deposition on Au S PR chip The deposition of graphene was notably pivotal in i nterfacing D NA oligonucleotides onto the S PR sensing system. We observed that t he drop -casting deposition of the FLG solution onto the Au sensing chip of the portable S PR sensor had favorably resulted in graphene with a few- layer struct ure. In Fig S1a, the optical microscopy figure showed a considerably uniform coverage of FLG on the Au layer, indicated by the homogenous transparent color spread onto the surface with tiny clumped particles over it. The surface morphologies visualized by FESEM in Fig S 2 demonstrates the FLG on top of the Au chip, indicated by some visible creases on the surface with contrast seen to the Au layer without graphene on top. The FLG structure is typically found in the drop-casting method as a result of the graphene film wrinkling due to the surface tension of water when the evaporation takes place during drying protocols [42]. It is notewor thy that the wrinkled structure of graphene is ascribed to the defects and holes in the lattice of tetrahedral sp3 hybridized carbon atoms providing extra bonds that finally form the atomically rough graphene sheets as compared to pristine graphene [43,44] . Raman detection of the FLG film in Fig S2 presents a substantial uniformity of graphene coverage by considerably similar spectral features taken at three different sites. The grap hene fingerprint peaks, such a s a visible 2D (~2680 cm -1), considerably 10 sharp G (~1571 cm-1), and an intense D (~1340 cm-1) peaks ascribing to the phonons coupling with two opposite wave vectors, sp2 carbon and sp3 carbon, respectively [45], were observed. The lesser intensity of 2D than the G peak indicates the few-layer graphene formation [46,47] , and the highly pronounced D peak (defects) , as well as small 2D peak, is attributed to the defects due to the removal of oxygen in the basal plane [45,48] . The rough graphene sheet would be beneficial in trapping the probe due to the high surface area in the next stage of surface functio nalization. This FLG solution drop-casting method holds a potency for a straightforward and brief process for graphene -based S PR biosensor development . Figure 2 is preferred in this location 3.2. Absorption profile s of GNu and GNr . The architectural building block of the gold nanostructures greatly determines its optical properties. The transmission electron micrograph (T EM) in Fig 2 inset shows that the GNu consists of poly -branch tips with an approximate tip -to-tip diameter of arou nd 70 -80 nm. The absorption spectr a of GNu and GNr in the PBS medium are demonstrated in Fig. 2. The GNu absorption appears optim ally at around 601 nm of wavelength. In the presence of ssDNA on the GNu following up the preparation of the probe , the absorpt ion peak was shifted to ~619 nm of wavelength. This finding is in good accordance with Rotz et al., 2015 who reported a similar range of wavelength shift s of gold nanostars spectrum after D NA conjugation with good maintenance of colloidal stability [49]. In contrast , as seen in the T EM image, in Fig 2 inset, the dimension of GNr has a length of ca. 40 nm and thickness of rod of about 10 nm. The GNr absorption showed two major peaks , with a maximum peak at 724 nm of wavelength and the minimum one at 522 nm. These peaks emerged as the consequence of dual orientations of the GNr , which has longi tudinal and transverse peak s [50,51] . In the presence of ssDNA, the minor and major peak s were blue- 11 shifted to 531 nm and 734 nm of wavelength, respectively , indicating a successful formation of ssDNA and GNr nanoprobes complex 3.3. Annealing -quenching protocol of Au sensing chip and GNPs effect s on the SPR signal enhancement The a nnealing and quenching step of the Au S PR sensing chip before graphene solution deposition was an attempt to enhance Au {111} orient ation on the surface [41]. Higher temperature annealing was sugge sted to obtain highly ordered Au {111}. However, considering that the melting point of the BK7 substrate is around 350 °C, the annealing point in our experiment was set up to 300 °C. Moreover, the quenching step with the exposure of the substrate to the co ld N 2 gas was conducted to avoid substrate crack , which easily occurs in the quenching techniques with cool liquid immersion . Song et al. reported that Au {111} film exhibited better binding absorption for graphene solution [23]. For the comparison purpose, we measured the probe immobilization on the FLG -covered Au S PR chip in the presence and absence of the annealing -quenching protocol , as displayed in Fig 3 a. The strong absorption and homogenous coverage of the FLG film onto the Au S PR sensing chip w ere assisted by the annealing -quenching protocols , which in comparison to the non -treated Au chip, dramatically amplified the S PR signal after the immobilization of GNP - DNA matrix via the π-π stacking force between graphene and ssDNA. The π -π stacking force exists in the mechanism of ssDNA immob ilization since both graphene and ssDNA shared identical characteristics as the electron -rich materials [16,23] . It is also essential to note the topological impact of the uniform F LG coverage on the Au sensing chip for probe capture. Pertaining to the convection -diffusion -reaction during the F LG drop-casting o n the Au chip , the convection -triggered evaporation of liquid and surface roughness of the graphene layer could 12 enable molecular absorption facilitating the D NA transfer to the graphene interface, which in turn cut off the diffusion time and yield high -sensitivity detec tion[24]. On the other hand , in the Au S PR chip without the annealing -quenching step, the probe immobilization stage yielded a negligible S PR signal. This is indicative of the poor FLG sheet coverage onto the Au chip. The FLG degradation was plausibly caused by buffer streaming in the reaction chamber creating the lack of Au {111} orientation with higher energy absorption of the graphene layer. This outcome is in a linear agreement with the report of Song et al. [23]. The morp hology of Au film in the presence and absence of the annealing is depicted in Fig. S3. Figure 3 is preferred in this location 3.4. Probe immobilization The gold nanorods (GNr) with the length of ~40-60 nm and aspect ratio 2.5 w ere applied to compare the SPR signal enhancement in the GNu -assisted probe immobilization stage . The S PR signal levels are presented in Fig. 3(b). The GNu -ssDNA performs three times higher S PR signal enhancement than the GNr-ssDNA probe ´s signal . This phenomenon is closely linked with the plasmonic absorption behavior of GNu and GNr , which likely occur in different wavelength region s. The plasmonic absorption of GNu is observed to be in the range of 600 -620 nm, which is correlated to the intensity peak of the OLED light source in this ex periment. Contrarily , the GNr maximum plasmonic absorption was observed in the range of 720 -750 nm wavelength , where the intensity of the OLED light source had dropped below the full -width half maximum (F WHM ) [39]. As displayed in Fig 3b, The GNu in this study had successfully provide d around three times higher enhancement i n the S PR signal than the GN r. 13 3.5 Detection of cssDNA target The probe D NA immobilization is pivotal in the overall study. In GN r-assisted D NA detection (Fig S 4a and Fig 3b), the S PR signal from the GNr was indicative of the probe D NA immobilization. However, compared to the GN u-assisted one shown in Fig 3a and 3b, the GNr signal is extremely low since the saturation level was achieved very rapidly within the low range level of S PR signal (Fig S 4a). In the detection of a series of css DNA concentration s, the SPR sensor detected the degradation of the signal level after the washing step by PBS solution in both GNr and GNu assisted -DNA detection (Fig S 4b and Fig 4a ), respectively. It is also notable that in both approaches, the S PR signa l dramatically leaped as the cssDNA target was injected into the reaction chamber due to the immediate change in the refractive index value resulted from the salt buffer from the target dilution. The D NA binding affinity is greatly affected by the io nic strength [52], and s alt mainly shows interactions with the negatively charged phosphate molecules [53]. The c ontrast was seen in the S PR signals after PBS wash in GN u-assisted DNA detection , which show s more pronounced signal reduction trends as higher cssDNA concentration was injected into the sensing chamber (Fig 4a ). The drop of SPR signal below the reference signal level (negative ΔSPR signal ) implies the release of ssDNA probes from the graphene surface by stronger hydrogen bond force during DNA hybridization [54] than the π -π stacking force between graphene and probe D NA [55–57]. The removal of the probe is related to the different binding energy where the D NA hybridization binding energy was estimated at around 65.7 kcal/mol (or equivalent to 2.85 eV)[35], while the π -π stacking force between ssDNA to the graphene surface is only approximately 0.49 to 0.61 eV[7,36]. The removal of the ssDNA and the GNPs away from the sensing area had resulted in a significant drop in the plasmonic field represented by a negative ΔSPR signal . This, by nature, structure , and morphology of the G NPs, was much stronger in the 14 GNu than GNr in providing the plasmonic effects . This phenomenon leads to the susceptible sensing method with the negative signals. Figure 4 is preferred in this location Fig 4b demonstrates the calibration plots f rom the level signal after PBS washing protocols in the real-time dynamic measurement from GN u and GNr-assisted D NA detection (Fig 4a and Fig S 4b). The GN u-assisted D NA detection yields a trendline with a correlation coefficient of 99% and an estimated limit of detection (L OD) based on the International Union of Pure and Appli ed Chemistry (IUPAC) definition using confidence level 3. It means th e LOD was calculated by the estimation of the concentration (the x -axis), when the signal level is reached 3 times of standard deviation of reference measurement , in correlation to the do se-response standard curve . The SD of the reference measurement was 0.23, consequently , the calculated LOD of ~24.5 fM cssDNA was achieved , significantly lower than that estimated in GNr SPR signal shown by 8.2 pM of cssDNA . Figure 5 is preferred in this location The calibration plot from the specificity test performed using non-complementary mssDNA target hybridization with the GNu -cssDNA probe was displayed in Fi g 4b. In this test, the hydrogen bond of the D NA hybridization was not supposed to ex ist, and hence, the mssDNA strand s were accumulated in the graphene layer due to the π -π stacking force. This mechanism produced a slight shifting of the delta SPR signals to the positive signal [58], which opposed the results from the specific binding of hybridized D NA. The combined calibration plo ts clearly distinguished the GNu paramount characteristics in providing S PR signal enhancement for both 15 highly sensitive and specific detection of D NA hybridization. The plasmonic field enhancement characteristics of GNu and GNr simulated in F DTD is presented in Fig. 5. The GNu showed prominent hotspots in every tip of its spicules , while the GNr hotspots were likely distributed in the edge of its diameter. The robust plasmonic profile of the GNu in the simulation affirms the findings on its UV -VIS absorption behavior shown in Fig 2. It is important to note two crucial contributions of t he GNu in D NA detection using our constructed S PR sensor. One, the surface roughness effects on the GNu facilitates higher probe D NA binding sites proven by the lar ger shift in the UV -VIS absorption outcomes than that in the GNr bonded -probe D NA Two, the sharp spicules and rough morphology of the GNu exhibits a drastic improvement of the SPR signals than the smooth -surfaced GNr shown in the F DTD simulation. Table 1 is preferred in this location Compared with the reported studies in related fields in Table 1, it is indicated that our modular and portable sensor using a graphene -based S PR sensor combined with GNu -assisted DNA detection shows excellen t advantages in terms of simplicity, portability, specificity. The detection limit in our proposed study is highly comparable to the commercial S PR, laboratory S PR as well as the S PR imaging system , which indicates a promising technique for D NA detection in a low concentr ation . 4. Conclusion The proof -of-concept of the DNA hybridization of MTBC , assisted by the plasmonic field from GNu has been presented in a portable graphene -based SPR sensor. The drop -casting of a few- layer graphene solution on the Au chip of the S PR sensor has paved the way for simple, time- 16 saving, and low-cost sensor production. The high accuracy of the detection reflected from the low detection limit and high specificity in D NA detection performed in this study offer new insights for early screenin g methods of bacterial infection, such as Tuberculosis infection in a rapid a low - cost manners with portable sensors possibly applied in a remote area or the third world countries as well as applicable for the use in the pandemic scenes. ACKNOWLEDGMENTS The authors wholeheartedly thank the Taiwan Ministry of Science and Technology for the research grant under contract number MOST 109-2221 -E-182-021-MY3 . B.A.P. acknowledges Chang Gung University for the research visit grant under BMRP741 . 17 Figures and captions Fig. 1 . Experimental configuration of D NA hybridization on a graphene -based SPR sensor . 18 Fig. 2 UV/VIS a bsorption spectra of GNu and GNr in the absence and presence of ssDNA probe . 19 (a) (b) Fig. 3. (a) The real-time measured SPR signal comparison of GNu -probe immobilization on the FLG layers deposited on Au chip with and without annealing treatment , (b). The comparison of SPR signal level from GNu -ssDNA and GNr -ssDNA probe immobilization on the FLG layer deposited on an annealed Au SPR chip 20 (a) (b) Fig. 4. (a) Real-time SPR sensor measurement of the series concentration s of cssDNA target hybridization with GNu -ssDNA probe, (b) The calibration plots showing the cssDNA target hybridization with GNu -ssDNA and GNr -ssDNA probes, and the specificity test from the hybridization of non -complementary mssDNA and GNu -ssDNA probe. 21 (a) (b) Fig. 5. The F DTD. simulation of plasmonic distribution on GNu and GNr. 22 Table and caption Table 1. Performance c omparisons of our work with other SPR -based DNA detection studies No Sensing platform Remarkable Technique Detection Target LOD. Ref. 1. Fiber optic SPR SAM -PEG surface chemistry binds the streptavidin for signal enhancement DNA -protein 2 nM [59] 2. SPR. DNA -templated polyaniline deposition DNA -PNA 5 nM [60] 3. SPR imaging Signal enhancement by colloidal Au NP DNA hybridization 10 pM [61] 4. SPR imaging Signal enhancement by Au NP MicroRNAs 10 fM [62] 5. Laboratory SPR GO, signal enhancement by AuNP DNA hybridization 10 fM [20] 6. Commercial S PR Sensia Graphene, signal enhancement by Au nanostar DNA Hybridization 0.5 fM [32] 7. Localized S PR. Au nanoisland, Signal enhancement by Au nanostar DNA hybridization 0.2–40 nM [63] 8. Localized S PR. Doxorubicin -modified gold nanoparticles for signal enhancement DNA hybridization 600 pM [64] 9. Commercial S PR Biacore X Signal enhancement by streptavidin MicroRNA 17 pM [65] 10. Commercial S PR Biacore X Signal enhancement by super - sandwich ass embly and streptavidin MicroRNA 9 pM [66] 11. Laboratory S PR. Surface -anchored rolling circle and AuNP amplification Tuberculosis DNA. 5 pM [67] 12. Modular and porta ble S PR. Few graphene layers, signal enhancement by Au nano urchin Tuberculosis DNA 24.5 fM This work 23