1. Introduction {#sec1}
===============

*Vibrio parahaemolyticus* (*V. parahaemolyticus*), a Gram-negative bacterium, is one of the important seafood-borne pathogens which has a serious impact on human health.^[@ref1]^ It is commonly found in coastal areas and marine products. Due to the widespread distribution in coastal and marine waters, *V. parahaemolyticus* causes Travelers' diarrhea in many countries including Thailand.^[@ref2]−[@ref9]^ To prevent food poisoning and assure food safety, it is very important to determine the contamination of *V. parahaemolyticus* in seafood products. Although culture-based biochemical methods have been widely used for identification and isolation of *V. parahaemolyticus*, this method requires laborious steps and is time-consuming. Recently, several sensitive and rapid detection methods for *V. parahaemolyticus* have been developed, such as polymerase chain reaction (PCR)^[@ref10]−[@ref13]^ and enzyme-linked immunosorbent assay (ELISA).^[@ref14]−[@ref16]^ However, these methods are still restricted by the need for skilled operators and expensive specialized equipment. Thus, simple, rapid, and sensitive methods for screening of *V. parahaemolyticus* contaminated foods are urgently needed to ensure food safety.

Magnetic separation (MS) technology is one of the most popular techniques for isolation and enrichment of bacteria.^[@ref17]−[@ref23]^ Compared with traditional separation techniques, magnetic separation has many advantages enabling bacteria to be quickly localized or retrieved with a common magnet. Using the magnetic nanoparticles (MNPs) modified with biological recognition elements, target bacteria can be easily isolated from complex components when an external magnetic field is applied. Among other biological recognition elements, a single-stranded DNA or RNA, called aptamer, is very promising in the development of bacterial separation and detection systems.^[@ref24]−[@ref33]^ Aptamers can bind to whole cells of target bacteria as well as antibodies with high affinity and specificity. The aptamers that are selected through *in vitro* selection or the systematic evolution of ligands by exponential enrichment (SELEX) possess many competitive advantages over antibodies, such as greater chemical stability, easy production, cost-effectiveness, and flexible modification. Although aptamer-functionalized magnetic nanoparticles (Apt-MNPs) for detection of pathogenic bacteria have been extensively reported, they have only been used for selective capture and enrichment of target bacterial cells from complicated samples. Quantitative detection of bacteria has been evaluated by additional signal generators such as fluorophores,^[@ref28]−[@ref30]^ enzymes,^[@ref31]−[@ref33]^ or other metals,^[@ref30]^ in which a signal amplification step and analytical instrumentation were required. Gold nanoparticle (AuNP)-based colorimetric assays have been extensively studied owing to the convenience of visual observation and simple operation. In this work, aptamer-conjugated magnetic nanoparticles (Apt-MNPs) in combination with AuNPs were utilized for intrinsic visual detection without signal amplification or extra instruments.

Here, we report a simple visual and sensitive colorimetric method for detection of *V. parahaemolyticus* employing aptamer-functionalized magnetic nanoparticles (Apt-MNPs) as a specific separator and aptamer-functionalized gold nanoparticles (Apt-AuNPs) as an indicator. The detection was designed on the concept of sandwich formation of Apt-MNPs and Apt-AuNPs due to the high affinity of aptamers to the corresponding bacteria. After magnetic separation, the detection signal was directly observed as a fading of the AuNP color. It should be highlighted that this concept can be evaluated by the naked eye and does not require an additional step for signal amplification as in other assays. The applicability of this method was also investigated in spiked raw shrimp samples and was compared with the conventional plate counting method.

2. Results and Discussion {#sec2}
=========================

2.1. Characteristics of COOH-Functionalized MNPs and Apt-MNPs {#sec2.1}
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The transmission electron microscopy (TEM) and attenuated total reflection-Fourier-transform infrared (ATR-FTIR) techniques were used to characterize the COOH-functionalized MNPs and Apt-MNPs. As displayed in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A, the ATR-FTIR spectrum of the COOH-functionalized MNPs showed a characteristic C=O stretching of the carboxyl group at 1701 cm^--1^. After aptamer conjugation, this peak disappeared while a new band at 1687 cm^--1^ corresponding to an amide bond was observed, indicating that the aptamers were successfully conjugated onto the surface of MNPs via a bond formation between the carboxyl groups of COOH-functionalized MNPs and the amino groups of amino-modified aptamers. The TEM image shows that the Apt-MNPs are spherical and the average size is approximately 10.6 ± 1.3 nm ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}B). The ζ-potential value of COOH-functionalized MNPs (−22.7 ± 2.3 mV) became more negative after aptamer conjugation (−30.9 ± 0.7 mV) due to the negative nature of DNA (−43.8 ± 0.8 mV) ([Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.0c01795/suppl_file/ao0c01795_si_001.pdf)).

![(A) ATR-FTIR spectra of COOH-functionalized MNPs and Apt-MNPs and (B) TEM image of Apt-MNPs.](ao0c01795_0002){#fig1}

2.2. Characteristics of AuNPs and Apt-AuNPs {#sec2.2}
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The morphology of AuNPs synthesized by citrate reduction is shown in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}A. The AuNPs are monodisperse and spherical in shape. The size distribution of AuNPs seems to be rather broad. As evaluated by SemAfore 5.21 from 125 random particles, the diameter was found to range from 8.8 to 24.6 nm giving an average of 15.2 ± 2.2 nm. The UV--vis spectrum of the AuNP suspensions (black line) showed a characteristic plasmon absorption peak at 524 nm and displayed the characteristic red wine color ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}B). The successful coupling of thiol-aptamers to AuNPs through Au--S bonding was confirmed by the shift of the absorption peak from 524 to 528 nm (red line). The Apt-AuNPs exhibited a lower ζ-potential value (−21.4 ± 1.9 mV) than the citrate reduced-AuNPs (−59.8 ± 3.2 mV) ([Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.0c01795/suppl_file/ao0c01795_si_001.pdf)). This change in the surface charge of the Apt-AuNPs is likely due to the replacement of citrate molecules on the AuNP surface by thiol-aptamers. The conjugation of the aptamer made the AuNPs very stable with the protection of the aptamer and preventing the Apt-AuNPs from aggregation in NaCl ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}). On the other hand, the unmodified AuNPs aggregated in the presence of NaCl with a color change from red wine to violet.

![(A) TEM image of AuNPs and (B) optical images and the corresponding UV--Vis absorption spectra of AuNPs (black line) and Apt-AuNPs (red line).](ao0c01795_0003){#fig2}

![Optical images and the corresponding UV--Vis absorption spectra of (a) AuNPs, (b) AuNPs + NaCl, and (c) Apt-AuNPs + NaCl.](ao0c01795_0004){#fig3}

2.3. *V. parahaemolyticus* Detection {#sec2.3}
------------------------------------

The detection assay was based on the use of aptamers as a specific recognition probe, MNPs as the capture material, and AuNPs as a signal detector. In the presence of target bacteria, aptamers on the surface of both MNPs and AuNPs bound with the target bacteria, leading to the sandwich formation of MNP--aptamer--bacteria--aptamer--AuNP complexes ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}). Then, the sandwich complexes were magnetically separated resulting in the fading of the suspension color. To prove this concept, the Apt-MNPs and Apt-AuNPs were incubated with *V. parahaemolyticus* cells and then magnetically separated. As shown in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}, the red wine color of the original AuNP suspension ([Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}a) faded significantly upon the presence of *V. parahaemolyticus* ([Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}c), whereas no obvious difference occurred in the absence of *V. parahaemolyticus* ([Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}b), indicating that the sandwich formation is the key for success.

![Proposed colorimetric method for *V. parahaemolyticus* detection using Apt-MNPs and Apt-AuNPs.](ao0c01795_0005){#fig4}

![Optical images and the corresponding UV--Vis absorption spectra of (a) Apt-AuNPs, (b) Apt-AuNPs + Apt-MNPs, and (c) Apt-AuNPs + Apt-MNPs + bacteria. (Concentration of *V. parahaemolyticus* was 10^4^ cfu/mL.).](ao0c01795_0006){#fig5}

To optimize the detection, the effect of the amount of Apt-MNPs and Apt-AuNPs was first studied. The results were evaluated in terms of the signal response (*A*~0~ -- *A*), where *A*~0~ and *A* are the absorbance of the supernatants in the absence and presence of *V. parahaemolyticus*, respectively. The signal response first increased with increasing Apt-MNP ([Figure [6](#fig6){ref-type="fig"}](#fig6){ref-type="fig"}A) or Apt-AuNP ([Figure [6](#fig6){ref-type="fig"}](#fig6){ref-type="fig"}B) concentration until a maximum was attained at 20 or 150 μL, respectively.

![Signal response (*A*~0~ -- *A*) as a function of the amount of (A) Apt-MNPs (Apt-AuNPs were fixed at 150 μL) and (B) Apt-AuNPs (Apt-MNPs were fixed at 20 μL). Concentration of *V. parahaemolyticus* was 10^4^ cfu/mL.](ao0c01795_0007){#fig6}

Upon utilizing the optimal amount of Apt-MNPs and Apt-AuNPs, the colorimetric detection of different concentrations of *V. parahaemolyticus* was investigated. [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}A shows that the absorbance of supernatants decreased with increasing *V. parahaemolyticus* concentration. A clear fading from red wine to colorless could be obviously differentiated by the naked eye (inset A). [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}B shows a good linear correlation between the signal response and *V. parahaemolyticus* concentration in the concentration ranging from 10 to 10^6^ cfu/mL. The linear equation of *V. parahaemolyticus* is *Y* = 0.0199*x* + 0.3075 (*R*^2^ = 0.9988). The limit of detection (LOD) is defined as the lowest concentration of bacteria that gives a signal 3 times higher than the standard deviation of the blank (3 SD~blank~/slope). Thus, the LOD for *V. parahaemolyticus* in the pure cultures was 2.4 cfu/mL. The limit of quantitation (LOQ) was 8.1 cfu/mL, which can be calculated from the 10 SD~blank~/slope.

![(A) Optical images and the corresponding absorption spectra of the supernatant after magnetic separation from the detection of different concentrations of *V. parahaemolyticus*. (B) Standard curve of the response signal (*A*~0~ -- *A*) versus the log concentration of *V. parahaemolyticus*.](ao0c01795_0008){#fig7}

To determine the selectivity of the assay based on the Apt-MNP and Apt-AuNP complexes, experiments were also performed to detect other pathogenic bacteria, including *Escherichia coli*, *Salmonella typhimurium*, and *Listeria monocytogenes* at a concentration of 10^4^ cfu/mL. As shown in [Figure [8](#fig8){ref-type="fig"}](#fig8){ref-type="fig"}, it is clearly seen that only *V. parahaemolyticus* induces a dramatic increase in the signal response, whereas other species produced signals as low as the blank control (in the absence of *V. parahaemolyticus*). These results clearly demonstrated that the developed method can specifically identify *V. parahaemolyticus*.

![Signal response (*A*~0~ -- *A*) upon detecting pathogenic bacterial strains. Concentration of bacteria was 10^4^ cfu/mL.](ao0c01795_0009){#fig8}

2.4. Detection of *V. parahaemolyticus* in Spiked Shrimp Samples {#sec2.4}
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To further validate the applicability and feasibility of this assay for detection of *V. parahaemolyticus*, raw shrimp purchased from a local market was used as a representative of the real sample. First, no *V. parahaemolyticus* was detected in the samples by the plate counting method. The shrimp samples were spiked with *V. parahaemolyticus* at a concentration of 1.0 × 10^4^ cfu/mL and detected using both the colorimetric method and plate counting method. Satisfactory recoveries were obtained, which is in agreement with data obtained from the plate counting method ([Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}). This verified the applicability of this method for detection of *V. parahaemolyticus* in real samples.

###### Quantification of *V. parahaemolyticus* in Spiked Shrimp Samples (*N* = 3)

  method                 original value (cfu/mL)   spiked concentration (cfu/mL)   measured concentration (cfu/mL), mean ± SD   recovery (%)   error (%)
  ---------------------- ------------------------- ------------------------------- -------------------------------------------- -------------- -----------
  colorimetric method    0                         1.0 × 10^4^                     1.10 × 10^4^ ± 0.04                          110.0          0.12
  culture-based method   0                         1.0 × 10^4^                     0.98 × 10^4^ ± 0.02                          98.0            

3. Conclusions {#sec3}
==============

A simple colorimetric assay for *V. parahaemolyticus* detection based on MNPs and AuNPs has been successfully developed. The complexation between MNPs and AuNPs induced by the target bacteria is the key for success. By employing the aptamer as a bacterial specific recognition element, this assay displayed excellent specificity and lower cost than antibody systems. The magnetic separation of MNP-Apt-bacteria-Apt-AuNP complexes from the mixture was demonstrated to be an excellent method of detection that could be seen as an obvious color change by the naked eye. This assay has the potential to be extended for detection of other foodborne pathogens or biological targets by changing the biological recognition elements.

4. Experimental Section {#sec4}
=======================

4.1. Materials {#sec4.1}
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FeCl~2~·4H~2~O, FeCl~3~·6H~2~O, HAuCl~4~, KCl, MgCl~2~·6H~2~O, NaCl, and ethanol were purchased from Sigma-Aldrich (Singapore). NaH~2~PO~4~·2H~2~O was obtained from Ajax Finechem (New Zealand). Thiosulfate-citrate-bile-salts-sucrose agar (TCBS Agar) was purchased from Difco (France). Trisodium citrate dihydrate and MgCl~2~·6H~2~O were obtained from Merck (Germany). Ammonia solution (28% w/v) and Na~2~HPO~4~ were purchased from QREC (New Zealand). *N*-(3-dimethylaminopropyl)-*N*′-ethylcarbodiimide hydrochloride (EDC) and *N*-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. The DNA aptamer having a sequence specific to *V. parahaemolyticus* was selected and identified by whole-bacterium SELEX, which was designed and reported by previously published research work.^[@ref28],[@ref31],[@ref33],[@ref34]^ The sequence of the *V. parahaemolyticus* aptamer is 5′-NH~2~-TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACT-3′ (NH~2~-aptamer) and 5′-SH-TCTAAAAATGGGCAAAAAACAGTGACTCGTTGAGATACT-3′ (SH-aptamer). The aptamers were synthesized by the Integrated and Technologies Company (Bangkok, Thailand). The binding buffer was prepared with 50 mM Tris--HCl, 5 mM KCl, 100 mM NaCl, and 1 mM MgCl~2~ at pH 7.4. Phosphate buffer solution was prepared with 0.1 M Na~2~HPO~4~ and 0.1 M NaH~2~PO~4~·2H~2~O.

4.2. Instrumentation {#sec4.2}
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The FT-IR spectra were recorded on a Perkin Elmer-Frontier ATR-FTIR spectrometer. The size and morphology of MNPs and AuNPs were examined by transmission electron microscopy (TEM) using a Philips Tecnai 20. The UV--Vis absorption spectra were measured with a spectrophotometer (Analytikjena, Specord 210 plus, Germany).

4.3. Bacterial Culture {#sec4.3}
----------------------

*V. parahaemolyticus* (ATCC 17802) was cultured overnight at 37 °C in alkaline peptone water (APW) with 1% NaCl (w/v) until past the logarithmic phase. The culture was then diluted with a binding buffer (50 mM Tris--HCl at pH 7.4, 5 mM KCl, 100 mM NaCl, and 1 mM MgCl~2~) until the OD~600nm~ = 0.5 (∼10^8^ cfu/mL). The bacterial culture was then serially diluted 10-fold from 10^6^ to 10 cfu/mL with the binding buffer. Plate counting was performed to verify the number of bacteria. The bacterial culture (100 μL) was diluted with a selective medium and coated on agar plates (TCBS agar) and cultured at 37 °C for 18 h to count colony-forming units (cfu).

4.4. Preparation of MNPs and Aptamer-Conjugated MNPs (Apt-MNPs) {#sec4.4}
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The carboxyl-functionalized MNPs were prepared by a one-pot coprecipitation method according to a previously reported method.^[@ref35]^ The conjugation of the aptamer onto carboxyl-functionalized MNPs was carried out via amide bond formation. The carboxyl groups on the surface of MNPs were first activated by EDC/NHS chemistry, allowing amine groups on the aptamer oligonucleotides to be covalently attached. Briefly, 250 μL of carboxyl-functionalized MNP colloidal solution (10 mg/mL) was added to a fresh mixture of EDC (50 mg/mL, 50 μL) and NHS (50 mg/mL, 50 μL). The solution was incubated at room temperature with gentle shaking for 30 min, followed by magnetic separation. The resultant MNPs were dispersed in 50 μL of DI water and 50 μL of NH~2~-aptamers (100 μM) was added and incubated for 45 min at room temperature with gentle shaking. Next, the Apt-MNPs were magnetically collected, rinsed with DI water, and suspended in 500 μL of DI water.

4.5. Preparation of AuNPs and Aptamer-Conjugated AuNPs (Apt-AuNPs) {#sec4.5}
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AuNPs were synthesized by the trisodium citrate reduction method.^[@ref36]^ A solution of HAuCl~4~ (0.42 mL, 48.17 mM) and DI water (9.58 mL) was boiled for 10 min with vigorous stirring. Then, trisodium citrate (1 mL, 57.80 mM) was rapidly added, stirred, and boiled for 15 min. The color of the suspension changed from yellow to red wine, which indicates that AuNPs were formed. The suspension was cooled to room temperature under continuous stirring. The concentration of the AuNP colloidal suspension was measured using the Beer--Lambert law from its absorbance at 524 nm and the extinction coefficient of 3.56 × 10^8^ M^--1^ cm^--1^.^[@ref37]^ The AuNP colloidal suspension was then diluted to 12 nM with DI water and stored at 4 °C.

The Apt-AuNPs were prepared according to the literature with some modification.^[@ref38]^ This protocol was based on the ligand exchange reaction between the thiol functionalized aptamer (SH-aptamer) and the citrate-capped AuNPs via the well-known gold--sulfur chemistry. The SH-aptamer (30 μL, 10 μM) was added to 600 μL of the AuNP colloidal suspension (12 nM). After allowing to stand for 24 h, the Apt-AuNP complex was aged with the addition of the phosphate buffer until a final concentration of 10 mM was reached. The suspension was allowed to stand for another 6 h and then 1 M NaCl was added to bring the total NaCl concentration to 0.1 M. The suspension was incubated for an additional 34 h at room temperature, followed by centrifugation for 30 min at 12 000 rpm to remove the excess oligonucleotides. After discarding the supernatant, the red pellets were washed, recentrifuged, and resuspended in 1 mL of binding buffer.

4.6. Detection of *V. parahaemolyticus* {#sec4.6}
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Bacterial samples (1 mL) were pelletized at 6000 rpm for 10 min and the supernatant was removed. Then, 150 μL of Apt-AuNPs (OD~524 nm~ = 0.5) and 20 μL of Apt-MNPs (1 mg/mL) were added to the pelletized bacteria and incubated for 45 min at room temperature with gentle shaking. After the magnetic separation, the samples were ready for detection. A color change was observed with the naked eye and the absorbance at 519 nm of the supernatant was read with a UV spectrophotometer.

4.7. Detection of *V. parahaemolyticus* in Spiked Shrimp Samples {#sec4.7}
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Shrimp samples were purchased from a local market. A 135 mL of the binding buffer was added to 15 g of the shrimp sample. The samples were shaken at room temperature for 10 min and centrifuged at 10 000 rpm for 5 min. The supernatant was subsequently filtered through a 0.45 μm membrane to remove particulate matter. *V. parahaemolyticus* at the desired concentrations (1.0 × 10^4^ cfu/mL) was then added to the samples for the experiments. The detection assay was similar to the procedures mentioned above.

The Supporting Information is available free of charge at [https://pubs.acs.org/doi/10.1021/acsomega.0c01795](https://pubs.acs.org/doi/10.1021/acsomega.0c01795?goto=supporting-info).ζ-Potential of DNA aptamers, MNPs, Apt-MNPs, AuNPs, and Apt-AuNPs (Figure S1) ([PDF](http://pubs.acs.org/doi/suppl/10.1021/acsomega.0c01795/suppl_file/ao0c01795_si_001.pdf))

Supplementary Material
======================

###### 

ao0c01795_si_001.pdf

The authors declare no competing financial interest.

This work was supported by the Thailand Research Fund (Grant No. MRG6180133), Thailand 4.0 project under Ageing Society Innovation Hubs, the Research Grant from Burapha University through the Sensor Innovation Research Unit (SIRU) for development and production of test kits, the Center of Excellence for Innovation in Chemistry (PERCH-CIC), and Science Innovation Facility, Faculty of Science, Burapha University. The authors thank Dr. Ron Beckett for English proofreading.
