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Solvent-sensitive nanoparticle-enhanced PCR assay for the detection of enterotoxigenic Escherichia coli

Stimulus-responsive nanoparticles are among the most utilized nanoscale materials in biomedical applications. As these nanoparticles exhibit a manipulable response to a particular stimulus, such as pH, heat, and organic solvent, they are potential signalling units in diagnostic assays. This study aims to enhance the limit of detection and reduce the turnaround time of magnetic nanoparticle polymerase chain reaction (PCR) enzyme-linked gene assay (MELGA), an advanced PCR-based technique termed the solvent-sensitive nanoparticle (SSNP)-enhanced PCR assay. This technique was proposed to detect pathogenic enterotoxigenic Escherichia coli (ETEC) through applying stimulus-responsive nanoparticles. The SSNPs were elaborated with three main components, including mesoporous silica nanoparticles as a structural unit, organic dye (Nile red) as a payload, and the corresponding organic solvent-sensitive polymer shell as “gatekeeper” (poly(maleic anhydride-alt-methyl vinyl ether, PMAMVE). A suitable organic solvent capable of inducing polymer swelling and dye dissolution was investigated by considering a solubility parameter. Using ethanol, the encapsulated Nile red can diffuse out of the SSNPs faster than other solvents and reach a constant concentration within 15 min. For the PCR inhibition study, various SSNPs concentrations (10–30 μg/reaction) were mixed with the ETEC gene and PCR reagent. The results showed that the particles in this concentration range did not inhibit PCR. By comparing the efficacy of conventional PCR, MELGA, and SSNP-enhanced PCR assay, the proposed technique showed a better detection limit than that of PCR, whereas that of MELGA was the lowest. Moreover, compared to MELGA or conventional PCR, this technique provided remarkably faster results in the postamplification process.

Bacterial cultivation and DNA extraction. Bacterial isolates of ETEC (DMSC 25093) were recovered onto tryptic soy agar (TSA) and multiplied in tryptic soy broth (TSB). The concentration of bacteria was adjusted by measuring the optical density value of 600 nm to 0.5, which is equivalent to 10 8 CFU/mL using a UV-vis spectrophotometer. The exact concentration of ETEC at OD 600nm was 1.41 × 10 8 CFU/mL. According to the instructions, bacterial DNA was extracted from the bacterial isolates using the Presto™ Mini gDNA Bacteria Kit. The bacterial DNA concentration was determined by measuring the optical density (260 nm) using a spectrophotometer. Nanoparticle characterization. The characteristics of the prepared SSNPs, including hydrodynamic size, polydispersity index (PDI), and zeta potential, were determined by Nanosizer. The morphology of the nanoparticles was analysed using TEM. Furthermore, the encapsulation efficiency of NR was determined by measuring the optical density of the supernatant after the synthesis process at 400 nm. Then, the values were calculated according to Eq. (1) and the standard curve.

Synthesis of dye-loaded solvent-sensitive nanoparticles (SSNPs
In addition, the SSNPs were tested for solvent response with various organic solvents. The SSNPs (1% w/v in DEPc-treated water, 500 µL) were mixed with organic solvents, including absolute ethanol, absolute methanol, acetone, ethyl acetate, DMSO (500 µL), and DI water (1 mL). The mixture was sonicated, mixed thoroughly for 0, 5, 10, 15, 20, 25, and 30 min, and subsequently spun down to separate the supernatant. The optical density of the supernatant of each fraction was analysed using a UV-vis spectrophotometer in scanning mode. The amount of NR released was determined based on the calibration curve.

Preparation of LT DNA primers immobilized onto the nanoparticles
Immobilization of LT-F onto MNPs. The specific LT forwards primer modified with an amine group at the 5′ end (5′-NH 2 -C 6 -GGC GAC AGA TTA TAC CGT-3′, NH 2 -LT-F) was immobilized onto carboxylated magnetic nanoparticles (COOH-MNPs) (1.45 wt%, hydrodynamic size 250 nm, zeta potential − 40 mV) through the carbodiimide method 20 . First, COOH-MNPs (1 mg) were washed twice with MES buffer (25 mM, pH 6.0, 500 µL). After that, NH 2 -LT-F (5 nmol in MES buffer) was added to the carboxylated MNPs and incubated at 900 rpm and room temperature for 30 min. Freshly prepared EDC solution (10 mg/mL, 10 µL) was subsequently added to the solution, and the mixture was then continuously mixed at 900 rpm overnight at room temperature. The residual concentration of unbound primers was measured from the supernatant after centrifugation using a UV-vis spectrophotometer. The binding efficiency and amount of immobilized NH 2 -LT-F on the carboxylated MNPs were calculated according to Eqs.  www.nature.com/scientificreports/ The LT-F-MNPs were then mixed with Tris buffer (50 mM tris(hydroxymethyl) aminomethane, pH 7.4, 500 µL) for 15 min at room temperature. Next, the nanoparticles were separated from the supernatant, resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0, 50 µL) to obtain the final concentration of stock primer (100 µM) and stored at 4 °C.

Immobilization of LT-R onto SSNPs.
Amino-modified specific LT reverse primer in MES buffer (pH 5.0) (NH 2 -LT-R, 5 nmol) (5′-NH 2 -C 6 -CGG TCT CTA TAT TCC CTG-3′) was added into the synthesized SSNPs (1 wt%) and then mixed at 900 rpm and 37 °C for 3 h. Next, the samples were centrifuged at 900 rpm for 15 min. The supernatant was then collected to evaluate the binding efficiency and amount of immobilized NH 2 -LT-R on the SSNPs using Eqs. (2) and (3) (where M of LT-R is 5596 g/mol). Afterwards, the primer-immobilized nanoparticles or LT-R-SSNPs were washed twice with Tris buffer at room temperature and 900 rpm for 15 min, resuspended in TE buffer (50 µL) to obtain the final concentration of stock primer (100 µM) and stored at 4 °C.
Conventional PCR. Conventional PCR was performed for limit of detection evaluation. A pair of primers were used to amplify a 450 bp segment of the heat-labile enterotoxin encoding gene (LT) of ETEC; these primers were designed using Primer-BLAST software and synthesized as previously reported 8,19 . Each sample was prepared in a mixture (50 µL) using 1 × Accustart II PCR Supermix (25 µL) containing 1.5 mM MgCl 2 , 0.2 mM dNTPs, 1.25 U of Taq DNA polymerase, 10 µM of each LT-F primer (5′-GGC GAC AGA TTA TAC CGT-3′) and LT-R primer (5′-CGG TCT CTA TAT TCC CTG-3′) (0.5 µL) and 25 ng/µL of DNA template (5 µL). DEPctreated water was used as the negative control. PCR amplification was conducted using a thermal cycler under optimal conditions. The process was started with a 2 min initial cycle at 94 °C and followed by 35 cycles of 94 °C for 50 s, 54 °C for 30 s and 72 °C for 30 s. A final extension step was performed at 72 °C for 5 min. The PCR products were then analysed for 450 bp segments by agarose gel electrophoresis at 100 V for 30 min and visualized using the Gel Doc™ EZ System after staining with GelRed.

PCR inhibition testing. PCR inhibition of LT gene amplification for the synthesized nanoparticles was
tested. Different amounts of SSNPs, i.e., 10, 15, 20, 25, 30 µg, and 0 µg (as a positive control), were added to each tube of the PCR mixtures, and a nonbacterial sample was used as a negative control. Afterwards, all samples underwent conventional PCR using LT-F primers (5′-GGC GAC AGA TTA TAC CGT-3′) and LT-R primers (5′-CGG TCT CTA TAT TCC CTG-3′) under the optimal conditions as described above and were then analysed using agarose gel electrophoresis.
MELGA. The MELGA technique was performed following the protocol of a previous study 8 . Each reaction was conducted in a mixture (50 µL) including 1 × Accustart II PCR Supermix (25 µL) containing 1.5 mM MgCl 2 , 0.2 µM dNTPs, 1.25 U of Taq DNA polymerase, LT-F-MNPs (1 µL), 10 µM biotinylated LT-R primer (biotin-5′-CGG TCT CTA TAT TCC CTG-3′) (LT-R-biotin) (1 µL) and 25 ng/µL ETEC DNA template (5 µL). MELGA proceeded under identical conditions for conventional PCR. The PCR product-bound MNPs (biotin-amplicon-MNPs) were captured by an external magnetic field, subsequently blocked with BSA solution (1% w/v in DEPctreated water) and then incubated with horseradish peroxidase conjugated streptavidin (SA-HRP) (0.1 µg/mL, 50 µL) in a dark chamber at room temperature for 60 min to allow biotin and streptavidin to interact. MELGA products (HRP-SA-biotin-amplicon-MNPs) were washed twice with DEPc-treated water and incubated with TMB substrate (3, 3′, 5, 5′-tetramethylbenzidine) in the dark at room temperature for 60 min to complete the oxidation of TMB. Finally, the absorbance of oxidized TMB was measured at 650 nm using a microplate reader and compared to a DNA-free sample as a negative control. The result was interpreted as the relative optical density (ROD) according to Eq. (4), where ODS and ODN are the sample and negative control, respectively.
Optimization of the SSNP-enhanced PCR assay. The SSNP-enhanced PCR assay was carried out based on the MELGA protocol using 10 µM LT-F-MNPs (5′-MNPs-NH 2 -C 6 -GGC GAC AGA TTA TAC CGT-3′) (1 µL) and various amounts of LT-R-SSNPs (SSNPs-5′-CGG TCT CTA TAT TCC CTG-3′) including 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 µL (equivalent to 10, 15, 20, 25, 30, 40 and 50 µg of SSNPs, respectively). For the postamplification step, the product bound onto MNPs (SSNPs-amplicon-MNPs) was sorted out using a magnet and was subsequently washed with BSA solution (1% w/v in DEPc-treated water), and the supernatant was discarded. Afterwards, a suitable organic solvent (200 µL) was added to the sample and subsequently mixed at 900 rpm for 15 min. After the supernatant from the samples was collected, the optical density was measured using a microplate reader with comparison to the negative control (DNA-free sample). The optical density values were calculated into the relative value (ROD) according to Eq. (4) 7 . The number of LT-R-SSNPs that provided the highest ROD was the best for the SSNP-enhanced PCR assay and was used in further experiments.

Results
Nanoparticle characterization. NH 2 -MSNs-NR-PMAMVE or SSNPs were used to measure the hydrodynamic size, size distribution, zeta potential, and encapsulation efficiency. According to the results in Table 1, the nanoparticles possessed a hydrodynamic size of 340.2 ± 3.4 nm with a narrow size distribution. The high absolute zeta potential value (> 30 mV) indicated that the nanoparticles exhibited good colloidal stability. Moreover, the morphology of the SSNPs was spherical (Fig. 2), as determined by TEM, and the particle size distribution was related to the results from dynamic light scattering. The particle size of the SSNPs in the dried state was 246.9 ± 28.6 nm, which was smaller than that determined using Nanosizer because PMAMVE shrinks in the dried state. Stimulation testing was performed with the SSNPs using various types of organic solvents, including ethanol, methanol, acetone, ethyl acetate, and DMSO; this was performed to identify the fastest stimulating solvent through which SSNPs delivers a relatively intense optical density. The obtained results are displayed in Fig. 3.

Sample
Hydrodynamic size (nm) Zeta potential (mV) PDI Encapsulation efficiency  www.nature.com/scientificreports/ After the SSNPs were incubated in ethanol, the highest optical density of the supernatant was obtained, suggesting that ethanol was the most efficient stimulus of these nanoparticles among the candidates. Afterwards, the time of stimulation to the endpoint was evaluated. The SSNPs were mixed with ethanol and DI water and subsequently shaken continuously for different durations (0, 5, 10, 15, 20, 25, and 30 min). Then, the optical density measurements were obtained at 553 nm for the supernatant of each mixture, as presented in Fig. 4.
All values were calculated as the accumulated concentration of NR released into the mixture. The results showed that after 10 min, the amount of NR released reached a stable point. The results indicated that the optical density of NR release can be evaluated after adding ethanol to the SSNPs and mixing for 15 min. PCR inhibition testing of SSNPs. The SSNPs are likely promising for this study due to their characteristics and response to the stimulus. Thus, the SSNPs in various amounts (0-30 µg/reaction) were further tested for compatibility with PCR while detecting the ETEC heat-labile enterotoxin gene. The results are displayed in Fig. 5.
The results showed that PCR inhibition was not detected in the presence of 10-30 µg of the SSNPs. This indicated that the SSNPs exhibited good compatibility with PCR.
Immobilization of LT primers onto nanoparticles. By using EDC as a coupling agent in the carbodiimide method, NH 2 -LT-F was successfully bonded onto the carboxylated MNPs through covalent bonds. Likewise, NH 2 -LT-R was covalently bound to the SSNPs (NH 2 -MSNs-NR-PMAMVE) through interaction between the PMAMVE molecule on the surface of SSNPs as a coupling agent itself and amino groups in NH 2 -LT-R. The primers exhibited a high binding efficiency to each nanoparticle, as shown in Table 2, indicating that most LT-F and LT-R were effectively immobilized on the carboxylated MNPs and SSNPs, respectively. The oligonucleotideimmobilized nanoparticles could therefore be used in PCR-based assays.   Fig. 6. According to the results, 3 µL or 30 µg of LT-R-SSNPs was considered the best amount for the SSNPs-enhanced PCR assay for the next experiments.

Discussion
Solvent-sensitive nanoparticles (SSNPs) composed of mesoporous silica nanoparticles (MSNs), Nile red (NR), and PMAMVE were synthesized as a structural support, signalling dye, and gatekeeper, respectively. PMAMVE copolymer is a good surface modification polymer that was reported to drastically improve particle stability and monodispersity; thus, PMAMVE contributes to successful amplification of the target DNA and is effectively used as a cross-linking agent 20 . The synthesized SSNPs with a hydrodynamic size of 340.2 ± 3.4 nm were accepted as homogeneous, as the PDI was less than 0.3. The high magnitude of the zeta potential value (− 47.5 ± 1.0 mV) of the nanoparticles suggested that the repulsive force was sufficient to maintain the physical colloidal stability upon storage. According to the results of the release profile, the optical density of the SSNPs stimulated in ethanol was highest. It was suggested that compared to other solvents used in the test, ethanol is the most effective stimulator with less than 15 min of stimulating time. A 15 min mixing period was the optimal time for inducing SSNPs to release the dye. When testing the inhibition of PCR by SSNPs, the sign of PCR inhibition was completely absent at 10 to 30 µg per reaction. Accordingly, the SSNPs were anticipated to be promising signalling support for postamplification analysis of a novel PCR-based assay. The newly developed assay, the SSNPs-enhanced PCR assay, was first optimized for an optimal amount of LT-R-SSNPs in PCR. The amount of LT-R-SSNPs ranged from 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 µL (equivalent to 10,15,20,25,30,40 and 50 µg of SSNPs). After amplification, all samples were subsequently treated with ethanol and mixed for 15 min before the optical density of the supernatant was measured. The result suggested that 30 µg of LT-R-SSNPs provided the highest positive result for the optical density, whereas 40 and 50 µg showed no significant positive result because the PCR was inhibited from an excessive amount of particles [20][21][22] . www.nature.com/scientificreports/ The detection limit of the three assays was evaluated for comparison. According to the results, the detection limit of MELGA appeared to be the lowest (10 -14 g/µL), followed by the SSNP-enhanced PCR assay and conventional PCR (10 -12 and 10 -11 g/µL, respectively). Compared to conventional PCR, the SSNP-enhanced PCR assay was found to be 10 times more sensitive, while MELGA was 100 times more sensitive than the SSNP-enhanced PCR assay. This result agreed with the positive results observed with the naked eye, as the colour changes in positive samples during the SSNP-enhanced PCR assay could hardly be differentiated from negative samples. Despite the slightly lower sensitivity, the SSNP-enhanced PCR assay could provide a remarkably faster turnaround time (approximately 20 min after amplification) than that of MELGA or conventional PCR, as summarized in Table 3. Moreover, the developed assay is easier to perform in practise, and before adding a stimulus to develop a www.nature.com/scientificreports/ colorimetric signal, only a single washing step is needed. Hence, the assay can prevent a loss in target amplicons, leading to a decrease in false-negative results. Moreover, regarding aerosol contamination during processing, this assay is safer for users since the final product is DNA, which is inactivated. Nevertheless, sensitivity and specificity evaluations must be performed in clinical samples to assess the true sensitivity, repeatability, and reproducibility values. Since NR dye can produce a fluorescent signal, the amplicons can be detected through fluorescent spectroscopy to enhance the sensitivity of this approach. Moreover, the simplicity and time of the assay possibly can be improved for use in the field by combining the DNA extraction and amplification steps.

Conclusion
Solvent-responsive nanoparticles (SSNPs) were synthesized, and these nanoparticles were composed of mesoporous silica nanoparticles (NH 2 -MSNs) loaded with Nile red (NR) and were coated with a solvent-sensitive shell poly(maleic anhydride-alt-methyl vinyl ether) (PMAMVE) as a gatekeeper (NH 2 -MSN-NR-PMAMVE). The nanoparticles were monodispersed and colloidally stable in aqueous medium. After adding the nanoparticles into PCR, the amount of the SSNPs (ranging from 10 to 30 µg/reaction) did not inhibit the amplification reaction, as a band at 450 bp corresponding to the molecular mass of the PCR product was clearly observed. The PMAMVE acted as a good gatekeeper to control the fast release of NR from the nanoparticles within 10 min; in addition, the carboxyl groups in the PMAMVE structure facilitated the covalent coupling of amino-modified specific LT reverse primer (NH 2 -LT-R) with high coupling efficiency (~ 97.8%). Using various concentrations of target ETEC DNA, the detection limit of the proposed SSNP-enhanced PCR assay (10 -12 g/µL) obtained was better than that of conventional PCR (10 -11 g/µL) but poorer than that of MELGA (10 -14 g/µL). Even though the limit of detection was higher for detecting ETEC than MELGA, the new assay possesses significantly faster postamplification analysis when compared to that of either conventional PCR or MELGA. The proposed assay reduced turnaround time by eliminating several washing, incubation and biotin-streptavidin interaction steps, resulting in a shorter turnaround time, less expensive reagents, and a simpler procedure in practice. Consequently, the SSNP-enhanced PCR assay can be a useful and promising PCR-based assay for detecting any target gene in the clinic.

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.