Ultrasensitive nano-gold labelled, duplex lateral flow immunochromatographic assay for early detection of sugarcane mosaic viruses

Sugarcane is one of the important food and bioenergy crops, cultivated all over the world except European continent. Like many other crops, sugarcane production and quality are hampered by various plant pathogens, among them viruses that infect systemically and cause severe impact to cane growth. The viruses are efficiently managed by their elimination through tissue culture combined with molecular diagnostics, which could detect virus titre often low at 10–12 g mL−1. To harmonize the virus diagnostics by molecular methods, we established a nanocatalysis-based high sensitive lateral flow immunochromatographic assay (LFIA) simultaneously to detect two major sugarcane viruses associated with mosaic disease in sugarcane. LFIA is known for poor sensitivity and stability with its signalling conjugates. However, we synthesized positively charged Cysteamine-gold nanoparticles and used them to prepare highly stable to sensitive immunoconjugates and as a colourimetric detection label. Further nanogold signal enhancement was performed on LFIA to obtain a high detection sensitivity, which is higher than the conventional immunoassays. The linear detection range of the nano-LIFA was 10–6 to 10–9 g mL−1, and with the signal enhancement, the LOD reached up to 10–12 g ml−1. This research paper provides relative merits and advancement on nano-LFIA for specific detection of sugarcane viruses in sugarcane for the first time.


Materials and methods
We used chemicals and materials essential for this research work from Sigma Aldrich and Merck Millipore with the highest purity. For the reagent preparation, nanoparticles dilution and other experimental works Ultrapure Milli-Q (Millipore-18 m Ω cm −1 ) water was used. All the glasswares (Borosil) needed for the nanoparticles processing was rinsed by Aqua regia (HCl: HNO 3 , 3:1) and thoroughly washed with Milli-Q water thrice, then ovendried before use. LFIA kit from mdi Advanced Microdevices, Ambala, India was used in the assays. Antibodies specific to SCSMV was earlier produced from our laboratory 38 , and antibodies specific to SCMV were provided by Dr Stephen Winter, DSMZ, Germany. For dot blot assays and ELISA, alkaline phosphatase conjugated secondary IgG (Sigma-Aldrich, Bengaluru), NBT/BCIP stock solution (Roche diagnostics), pNPP (Nitrophenyl phosphate disodium salt from Sigma) were used.

Virus sample extraction and purification. Healthy and virus infected sugarcane leaf samples (varieties
Co 976, Co 7706 and CoC 671) collected from ICAR-SBI farm. The samples were confirmed for virus infection by RT-PCR assays with coat-protein gene specific primers 39,40 . Plant serum was prepared by homogenizing one gram of leaf tissue in the presence of liquid nitrogen with the sterilized ceramic mortar and pestle. Further the homogenate was pulverized with 10 or 20 volumes of extraction buffer [0.02 M phosphate buffer, 0.15 M NaCl, 0.02% Sodium azide, pH 7.4, 0.05% Tween20 and 2% PVP (40,000 MW)] and then filtered through two layers of mira or muslin cloth. Highly positive (isolate Co 7706) sample was used to purify the virus protein to use as a standard for this assay 41 . The purified virus protein quantified as 13.4 mg/ml by Nanodrop spectrophotometer (Thermo Scientific) at 260/280 nm.

Synthesis of Cys-AuNPs.
There are no standard protocols in the literature that enable direct preparation of cysteamine capped cationic nanoparticles for the specific purpose of antibody conjugation. Therefore, we adopted a protocol described elsewhere 42,43 and modified the protocols to obtain stable cationic spherical gold nanoparticles with a narrow size distribution of 10-20 nm. Brought an aqueous solution of 40 ml HAuCl 4 (1.4 mM) into a clean flask at room temperature and gradually added the aqueous solution of 400 μl (213 mM) of Cysteamine hydrochloride (C 2 H 7 NS) onto the solution under vigorous stirring. Twenty minutes later, a freshly prepared Sodium borohydride (NaBH 4 ) of 40 μl (10 mM) was directly dropped into the reaction mixtures at the same stirring condition. The following 8-10 min was crucial to obtain the controlled synthesis of gold nanoparticles (AuNPs) in solution, also indicated by an intense wine red colour. The synthesized gold nanoparticles also rendered the striking red colour impression on the glass flask (anionic silicate ions), probably due to the attraction of cationic charged particles. Further, cysteamine stabilized gold particles were subjected to the standard characterization methods such as transmission electron microscopy (TEM), UV-Vis spectroscopy, zeta analysis for the size (d.m) and surface charge.
Preparation of nanogold-immunoconjugates. The detection sensitivity of polyclonal antibody (pAbs) specific to the SCSMV and SCMV was initially evaluated by ELISA and dot blot assay (1:100-1:500 dilution) on different virus isolates infecting sugarcane. Before performing the conjugation, glycerol in the pAbs solutions was removed by Amicon 10 kDa centrifugal filter with the choice of PBS buffer. Both SCSMV and SCMV Cys-AuNP-conjugates were prepared separately. An amount of 50 µg mL antibody diluted in 0.01 M PBS containing 0.02% sodium azide was added directly to 1 ml of Cys-AuNPs (OD 530 = 1.8), and the suspensions were vortex mixed for one hour at 25˚C; after that, BSA and sucrose were added to a final concentration of 0.2% and 2% (w/v) respectively and again vortex mixed for 30 min. Meanwhile, the simple "salt challenge" method was performed by adding 1 M NaCl with nanogold-antibody conjugates to confirm their surface coverage, which must be protected from the salt-induced aggregation 23 . Subsequently, the spectra of the nanogold-labelled conjugates were recorded with UV-Vis absorbance spectroscopy.
Preparation of LFIA. The LFIA strip (refer graphical abstract) comprised of four components: a sample pad, a conjugate pad, Nitrocellulose membrane (NCM, pore size 8 μm), and an absorbent pad. All the membranes were sourced from a commercially available LFIA preparation kit. The kit provided different membrane types needs to be pre-selected based on the flow time and flow pattern. High sensitivity was obtained in smaller pore sized NCM (8 μm), but wicking time was comparatively more extended than the larger pore sized NCM. For faster results and high affinity, a larger pore size of NCM was the best choice. Finally, the assay performance might be vary for complexity of samples and conjugate label used. Before assembly of pads, conjugate pad pretreated with PBS buffer (pH 7.4) containing 2% BSA, 2% PVP, 1% sucrose and 0.25% Tween 20 and allowed to completely dry at 37 °C for 2 h or RT for 24 h. This combination of buffer components was new and we tried especially for a thorough release of Cys-AuNPs-conjugates from the conjugate pad. Then, the desired volume of Cys-AuNPs-conjugates (OD 1 at 530 nm) was directly dispensed by micro pipetting onto a pre-treated conjugate pad (width of 6 mm) at a single instance. The conjugate pad was allowed to dry at RT for 4-5 h to prevent the humidity before assembling. Next, the nitrocellulose pad was used to immobilize antibodies in the test and control line. The blocking buffer (0.1% BSA in PBS) diluted capture antibody [(1.0 mg/mL) (SCSMV or SCMV)] and rabbit IgG-secondary antibody (1 mg/mL, 1:100, 200) loaded on the two printing channels in mdi membrane printing workstation and printed as test line (T) and control line (C) on the NC membrane (4 mm gap) at a medium speed, and then dried at 37 °C for four h. The sample pad is often treated with saline-sodium citrate buffer (SSC 4x) for human samples, however for plant samples no such pre-treatment is required since it was already filtered through mira cloth. Thus prepared conjugate pad adhered and then sample pad (half-width overlapped) on the top of antibody printed NC pad, after which attached the wicking pad at the bottom of NC pad. After all, assembled in the backing card, the plate was then cut into 4 mm wide strips by a strip cutter or manually by scissors. The prepared test strips were stored in a drying oven for a few minutes, then backed and sealed in the aluminium pouch with a desiccant. Duplex LFIA preparation. Two conjugation pads were prepared separately with SCSMV and SCMV nanogold-conjugates for duplex detection assay and placed together (overlapped) in a single strip. Conjugate labels were subjected to mild sonication before loading onto the pads. As mentioned earlier, two test lines were printed (0.5 cm gap) on the NC membrane with the SCSMV and SCMV diluted (0.5 mg/ml) antibodies.
Optimization and detection of LFIA. Principally, the LFIA performance was demonstrated by adding a series of diluted (1 × PBS) purified virus proteins (SCSMV) [10 µg/ml to 10 pg/ml]. The intensity of the test line was then image captured to establish a standard calibration curve. For that, 30 µl of the purified virus protein sample was dispensed on a sample pad, and allowed to react with AuNP-immunoconjugates for one minute (pre-incubation), then 50 µl of 1 × PBS was added to the sample pad to flow the conjugate contents towards the wicking pad. Pre-incubation might help to improve the detection sensitivity as well. Generally, the visibility of the test line and control line is defined as a positive, and the absence of a visible test line define as negative. With the purified protein sample, the absence of signal was recorded as ultra-low signal. "The presence or absence of a control line respective to the assay is correct or false". Similarly, the duplex assay was performed with the standard virus protein dilutions, and resulting images were captured for further analysis. Relative sensitivity of this LFIA was also determined through the standard Dot blot assay with SCSMV (1:500) antiserum (96 well Bio-Dot, www.nature.com/scientificreports/ Biorad) with alkaline phosphatase enzyme conjugates (1:2000) and NBT/BCIP substrates 44 . The output of colour intensity was image captured and measured to correlate with the enhanced nano-LFIA results.
LFIA with citrate-AuNPs versus cysteamine-AuNPs. The comparative sensitivity of one-week-old LFIA strips imbedded with the conjugates of Citrate-AuNPs and Cysteamine-AuNPs was tested using two-fold diluted purified SCSMV, and the intensity of test and control line images were recorded. Citrate-AuNPs synthesized by standard procedure and antibodies were conjugated followed by the protocol described elsewhere 27,45 . The average size of the Cit-AuNP obtained was 30 nm in diameter, spherical in shape, and the surface charge was usual electronegative (data not shown). The size and shape of both AuNPs were much similar, and major variation occured in their surface properties. Significantly, the surface properties are the directing factor that controls the orientation of antibodies on the AuNPs. To verify this statement, more solid evidences were generated through the novel immunoassay testing of both conjugate-AuNPs with secondary IgG-conjugated with enzyme alkaline phosphatase (AP). The mechanism behind the test was how much AuNPs-conjugated-primary-IgG-(Fc region) remain accessible to the secondary-IgG (Fab region) (refer the illustration at Fig. a). Taken 1 ml of both Cit-AuNPs and Cys-AuNPs-immunoconjugates with the addition of 1:1000 diluted AP-conjugated-secondary-IgG, kept at room temperature for 1 h. Later, the solutions were centrifuged (12,000 rpm) to remove unbound secondary-IgG, and the pellets were redispersed to the final volume of 1 ml by Milli-Q. Further, dispersed the solutions (100 µl) into a 96well plate and each well was loaded with 100 µl of pNPP substrate to react with APconjugated-secondary-IgG. For this test, positive control was used with primary-IgG (1:1000) incubated with secondary-IgG-AP and water plus secondary-IgG-AP used as a negative control. Recorded the final colorimetric signal/OD value by using an ELISA reader at 405 nm (SpectraMax plus 384, Molecular devices, USA).
Ultrasensitive detection by signal enhancement. All the standard dilutions that produced characteristic signals in LFIA were taken to the signal amplification procedure. Gold-enhancement solutions prepared as described elsewhere 46 . Briefly, solution 1: 2.5 mM HAuCl 4 (gold chloride) was prepared at 10 mM MES buffer (pH 6), adjusted using 4 M NaOH solution. Solution 2: 1.027 M H 2 O 2 prepared in 10 mM MES buffer 6. An equal volume of solutions 1 and 2 were mixed and used during the enhancement procedure. Before doing enhancement procedure, the NC pad was washed twice with washing buffer and dried to reduce background signals. For this assay, NC pad with test and control zones from the resulting strips separated by cutting and used to immerse in the instantly pooled gold enhancement solution 1 and 2 precisely for 2 min. The reaction was stopped immediately by dipping into DDH 2 O. Once the NC pad is immersed in the enhancement solution, the autocatalytic reaction is initiated to deposit the gold ions over GNPs, which increases the size of particles and gives dark blue colour in the test zones. Now, the test zones are visible to the naked eye and made easy for quantification by determining intensity level. The total events of gold nanoparticle hybridization detection to signal enhancement were captured in Scanning electron microscopy and presented here.
In-field test. Nearly  Interpretation and distribution of data. All the data compared with a standard and controls were analyzed by the standard statistical procedures. The experiments was repeated wherever necessary to minimize random errors and achieve statistical significance: MS Excel and Origin 8 pro software are used to organize and analyse data.

Research involving plants.
Our experimental research with plants was carried out fully complying with our Institutional and International guidelines-the collection of samples, safe-handling and disposing performed within our Institute premises.

Results and discussion
Physiochemical characteristic features of Cys-AuNPs. The optical emission intensity (due to high monodispersity) of Cysteamine stabilized gold nanoparticles was comparatively brighter and more intense than Citrate capped gold nanoparticles that may help the enhanced colorimetric detection signal in the paper sensing platform (Fig. 1b). The colloidal solutions taken during particle synthesis (1-10 min) have shown the evidence of gradual reduction of auric ions (Au 3+ -A 0 ) to nanoscale gold particles (AuNPs) under the analysis of UV-Vis absorption spectroscopy (Fig. 1a). The maximum (λmax) and strong absorbance peak of 520-540 nm was observed for the reaction solution taken at 10 min synthesis which is corresponding to the monodispersed colloidal gold nanoparticles (AuNPs) in the solution. It was seed mediated growth of AuNPs, principally initiated by thiol in the cysteamine and subsequently stabilized by the strong reducing agent of sodium borohydrate. Thiol is a highly attractive to metal ions involved in the nanogold seed synthesis further stabilized by borate ions. Average size of the Cys-AuNPs was determined as 24 nm (dm) through TEM and DLS and the shape was observed like spherical in the same image (Figs. 1a & inset, 2a). TEM image showed well dispersed nanoparticles by the cysteamine capping agent and borate stabilizing agent. We have had geometrically similar particles, con- www.nature.com/scientificreports/ sistently after many replications of synthesis that is a profound characteristic feature of Cys-AuNPs. The results of Zeta potential (mV) analysis for the Cys-AuNPs revealed that the electric potential and the surface charge of gold nanoparticle predicted as highly positive charged to the near value of + 30 mV in the graph and found some degree of measurable electronegativity in the same because of the borate (Fig. 2c). Zeta potential values of nanoparticles less than + 25 mV or greater than − 25 mV typically have low degrees of stability and dispersity in solution was reported in earlier study 47 . Consent with this statement, Cys-AuNPs (greater than 25 mV) in a storage glass bottles has shown good stability in months at 4ºC but not at the RT. Eventually, the behaviour of Cys-AuNPs at different thermal conditions incubated for 24 h showed greater stability at 4, 10, 20, 30-35 and 40 °C. As shown in Fig. 3a, no major distinct variation in the UV-Vis absorbance spectra was observed at the abovementioned thermal conditions. Whereas when storage temperature was below 4 °C or freezing state (− 20 °C), particles were reverse aggregated by changing their colour to a bluish solution, and no absorbance peak was observed. Also, the solution exposed to light irradiation showed decreased absorbance maximum meant for the growth of particles in the solution due to the free-energy absorbance from photons.
Cys-AuNPs as potential diagnostic-immunolabel. The functionalization of AuNPs with antibodies has been most important criteria and cumbersome process for successful immuno-sensing assays. Cysteamine (2-mercaptoethylamine) is a known organic compound consist of two functional groups such as amine and thiol which can be a potential coupling molecule to the antibodies 48 . However, carboxylate and sulfhydryl, consist in the antibodies are the most targeted and favourable conjugation sites for bioassays 49 . With the Cysteamine stabilized AuNPs, the presence of these two reactive binding sites were evidenced by the presence of positive charge (corresponding to amine) and modest negative charge (corresponding to thiol and borate) in the Zeta potential analysis was earlier discussed (Fig. 2c,d) 50 . Here, Cys-AuNPs might offer two strong interactions of ionic and covalent between the carboxyl tail and sulphur groups (through chemisorption) to the antibody, which are predominantly present in the Fc region (Fragment crystallizable region) and not in the antibody-binding site 51 . Combined ionic and covalent bonding energy of the two ensured specific and ideally oriented immobilization of antibodies on AuNPs. Therefore, the antigen binding (Fab) sites become well accessible for binding with their virus antigens. Hence, promisingly, the cysteamine stabilized AuNPs could offer combination of a strong coupling strategy (ionic and disulfide) for a stable conjugation of antibodies on them and this was also evidenced in the DLS measurement (Fig. 2b). This feature might help to improve the virus antigen detection capability and sensitivity when compared to other custom used AuNPs conjugation methods. Further, for the confirmation, we have performed Fourier transform infrared spectroscopy (FTIR) to analyze the bonding and surface characteristics between the cysteamine and Cys-AuNPs. The overlaid infrared spectrum taken (model Shimadzu FT/IR-6800typeA) with standard calibration and procedure showed deformed spectral peak obtained at the 2900-2700 cm −1 corresponding to B-H stretch (boranes), which attributed to a reduced state of sodium borohydride, the well-known strong reducing compound involved in the AuNPs synthesis (Suppl. Figure 1). There was no reference alone for sodium borohydride executed. The IR spectra of cysteamine (bottom), exhibited two main stretches of high absorption peak dedicated to primary (N-H) amine (1592 cm −1 ) and sulfhydryl (-SH) groups (470 cm −1 ) with standard frequencies. To the connection, in the upper IR spectra, the variation with lowintensity peaks at high frequencies observed in Cys-AuNPs shifted the wave number 1592 cm −1 to 1658 cm −1 and 470 cm −1 to 676 cm −1 attributed to open chain amino group and disulfides (S-S stretch). Generally, thiols participate in a thiol-disulfide exchange or coupling of two thiol groups by the oxidation results the disulfide www.nature.com/scientificreports/  www.nature.com/scientificreports/ bridge (Suppl. Figure 1) 52 . Hence, we concluded that cysteamine has given active sites by the interconversion (oxidation) of both a primary amine and thiol molecules in the AuNPs synthesis process. FTIR results have very well correlated with the results discussed in this section earlier. Further, we observed the overlaid peak of 3014-2943 cm −1 was the characteristic broad feature of C-H stretching region present in the cysteamine which might be part of stabilization of Cys-AuNPs along with the boranes 53 . Storage temperature is another factor that influences the lateral flow assay sensitivity which is absolutely associated with antibody conjugate labels 54 . The structural and chemical stability of antibodies based on hydrogen bonds which are more stable at low temperature. According to our analysis, cysteamine capping produced more compatible binding to the antibodies, thus resulted no aggregation of nanogold immunoconjugates upto 60 days at 4 °C (Fig. 3b). Moreover, the detection sensitivity of stored conjugates was similar compared with freshly prepared conjugates.
Performance of LFIA. "LFIA"-based biosensing depends on the colourimetric signals originating from the gold nanoparticle hybridisation conjugated probes with target sugarcane viruses. LFIAs are easy to operate because the results can be acquired in a few minutes and are usually binary as positive or negative. In our LFIA, purified virus protein at the linear concentration of 10 µg to 10 ng showed a good detection sensitivity without the gold enhancement (Fig. 4a). The intensity of nanogold hybridized signal on the NCM was in high visibility to low visibility correlated with the linear concentration of virus protein tested. While the concentration close to 1 ng and below was not offered any visible signal in the T line. However, after placing the membranes into gold enhancement solution, the intensity of the T line signals was enhanced tenfold from the original signal, and the visibility improved at all the concentrations, including for the lowest concentration of 1 ng-10 pg (Fig. 4b).
From the results of standard curve analysis, it was determined that the system of nanogold signal enhancement improved the sensitivity of sugarcane virus detection up to 10 -12 g mL −1 . The single-plex assay was effective as it responded well to all the sample concentrations. Control lines were visible in all the strips, and the intensity varied upon the hybridization event of the T line and with the flow rate of conjugate labels. From the signal enhancement results, a standard curve was generated with the polynomial fit, which showed a correlation among the various concentrations of tested virus protein, which could help to find the unknown concentration of virus, to be tested (Fig. 4c). SEM images taken with the test strips showed a series of differential occurrence of nanogold conjugates on the NCM before and after hybridization in the T line and then tenfold enlarged size particles after the application of gold enhancement solution (Fig. 5a-c). Moreover, the uniformity of enlarged size particles observed in the SEM images was clear evidence of controlled signal enhancement resulting from the 2 min precise incubation with the gold enhancement solution (Fig. 5c). Inappropriate incubation time with enhancement solution might cause the variation in the signal intensity from one set to another.
Comparative sensitivity (Cit-AuNPs Vs Cys-AuNPs). As briefly pointed out earlier, one-week-old citrate-AuNPs conjugates showed poor detection sensitivity in the lateral flow membrane at all the tested twofold serially diluted purified SCSMV. In contrast, Cys-AuNPs conjugates in the lateral flow membrane showed improved sensitivity after one week of storage at 4 °C (Fig. 6a-c). Those images of LFIA indicate the differences of intensity in both T and C lines between the Cit-AuNPs and Cys-AuNPs. The calculated (Fig. 6c) detection sensitivity of Cys-AuNPs was tenfold higher than the Cit-AuNPs at the lowest virus concentration. Both gold nanoparticle geometries were similar; the surface chemistry alone played a critical role in proving longevity and sensitivity. The results of comparative sensitivity were apparent evidence for the difference between covalent conjugation of Cys-AuNPs and simple physisorption of Cit-AuNPs to the antibodies. This statement was demonstrated clearly with the proof from our antibody orientation study (Fig. 7a,b). From Fig. 7b, colourimetric    www.nature.com/scientificreports/ OD values show the perfect distinction between the Cys-AuNPs and Cit-AuNPs-immunoconjugates reaction towards the secondary-IgG-AP. Compared to the positive control, a negligible amount of colourimetric signal was recorded for Cys-AuNPs-immunoconjugates. It denoted only the insignificant amount of the Fc region exposed to the solution and the remaining SCSMV antibodies oriented perfectly to Cys-AuNPs in an "end-on" position. Meanwhile, Cit-AuNPs showed almost fivefold higher than Cys-AuNPs and twofold lower than the positive control. It was evident that there was less possibility of perfect antibody orientation to Cit-AuNPs, which could be like Head/side-on position 55 . These results were the evidence of the stability of nanoparticle conjugates which were reinstated by the antibodies bonding and their steric hindrance to gold nanoparticles further improved the stability.
Performance of duplex-LFIA assay. The experimental results revealed that duplex assay could detect two important sugarcane viruses that predominantly coexisted in many important sugarcane cultivars. Duplex LFIA is never known for any plant virus detection. Also, embedding two conjugated reaction pads on one strip seems to be successful in our studies. We evaluated the efficacy of our duplex assay tested with four samples, and we observed slightly visible test lines of T1 & T2 in all the strips, corresponding to SCSMV and SCMV (Fig. 8a) ( Table 1). Distribution of both viruses in one sample gives a lower intensity response in the two test lines as compared to the single-plex assay. Further, the poor detection signal was improved to tenfold after applying the gold enhancement solution, which facilitates clear visibility of both test lines and control lines (Fig. 8b). The enhanced signal intensity range was calculated and interpreted with the standard chart that showed the LOD of duplex assay between the ranges of 10 ng and 1 ng both viruses (Fig. 8c). This duplex assay also determined that the SCMV titre was a little higher than the SCSMV in the infected sample, as evidenced in the bar diagram (Fig. 8c).
On-field diagnosis. Among the 10 samples tested in the nano-LFIA (Table 1), six samples were initially identified as positive for SCSMV with a visible low-intensity signal at T Lines (Suppl. Figure 2). The remaining four T lines corresponding to SCMV were subjected to gold enhancement application and found two samples were positive, and the last two were found negative. We found that the LFIA results 100% matched to RT-PCR analysis. The on-field diagnosis required 7-10 min to determine whether the samples were positive or negative with the ultrasensitive detection range of 10 ng to 1 pg. Nevertheless, the extraction of virus samples to be tested extended the overall testing time. Henceforth, we tried the juices extracted from virus-infected sugarcanes, and the results were found satisfactory with the help of a four-minute signal enhancement application. Two min preincubation of samples at conjugates pad found to give improved signal in the conventional LFIA. The specificity of our nano-LFIA tested with other sugarcane infecting viruses of sugarcane yellow leaf virus (ScYLV) and sugarcane bacilliform virus (SCBV) and found 99% accuracy in the specificity only for SCSMV and SCMV (Data not shown).
Relative sensitivity. To estimate the relative sensitivity, statistical significance was generated from the results of dot-blot immunoassay with the variable purified virus concentration as mentioned earlier (Suppl. Figure 3). The estimated relative sensitivity between nano-enhanced-LFIA (Y-axis) and dot-blot assay (X-axis) found a moderate positive correlation, which means there is a tendency for high X variable scores to go with  www.nature.com/scientificreports/ high Y variable scores (and vice versa) (Fig. 9). Briefly, nano-LFIA has shown an increased tendency in detecting variable virus concentrations. In contrast, the dot-blot assay showed poor sensitivity to the varying concentrations, which doesn't statistically correlate with the nano-LFIA detection sensitivity. Hence it is assumed that nano-LFIA has a good linear sensitivity from the higher to lower virus titre than dot-blot assay.
Summary. The low sensitivity and stability are the primary factors that prevent LFIA become an industrystandard diagnostics. Many researchers developed LFIA with increased sensitivity through much-distinguished modification such as various formats, different types of pads, different biorecognition and sensing labels than detection systems [56][57][58][59][60] . Likewise, we developed an LFIA with significant modification with conjugate tags that performed as a dynamic sensing label compared to a frequently used Cit-AuNPs. The signal enhancement system with gold nanoparticles is another excellent nanocatalytic method commonly used in LFIA and other gold nanoparticle-based sensors. Such a method is developed for the first time for sugarcane viruses with the linear detection sensitivity range of 10 -6 to 10 -12 , almost equivalent to RT-PCR detection range. Moreover, increasing the incubation time of test lines at the enhancement solution may further improve the detection signal to 20-fold and more. However, it must be precisely applied to all the strips to be tested. This assay helps early and rapid diagnosis of SCSMV and SCMV in sugarcane seed materials where virus titre is often in the picomolar concentration. If LFIA is developed with monoclonal antibodies, the LOD will be on the scale of femtomole concentration. In addition, an approach of 2 min pre-incubation of the sample with conjugate labels in the conjugation pad could increase the sensitivity of the conventional LFIA 61 . Since, LFIA functioned in multipad components, the selection of pads are also the critical limiting factor for any successful LFIA development. Sensitive diagnostic assays are not applied for sugarcane diseases due to higher costs and a technical workforce with laboratory support. Hence, gold nanoparticle-based LFIA assay is economical and affordable for sugar industry and seed cane farmers in a county like India. It is also a practically feasible diagnostic assay for diagnosing sugarcane viruses and probably has a commercial value in the billion-dollar sugarcane industry (Fig. 10). www.nature.com/scientificreports/

Data availability
All data analyzed during this study are included in this published article (and its Supplementary Information files). www.nature.com/scientificreports/