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Bacillus subtilis DNA fluorescent sensors based on hybrid MoS2 nanosheets [1]

['Son Hai Nguyen', 'School Of Mechanical Engineering', 'Hanoi University Of Science', 'Technology', 'Hanoi', 'Van-Nhat Nguyen', 'College Of Engineering', 'Computer Science', 'Vinuniversity', 'Mai Thi Tran']

Date: 2024-02 

(A) The SEM image captured by HITACHI-S4800 confirms the nanosheet morphology (inside the green region of interest); the contrast was enhanced. (B) The XRD pattern obtained by Rigaku MiniFlex600, and (C) The absorbance and photoluminescence attributes. Absorbance (blue line) was determined using a DeNovix UV-Visible spectrometer (Model: DS-11 FX+). The fluorescence intensities (orange line) were recorded using a spectrophotometer with a 10 nm slit-width (SpectraPro HRS-300, Teledyne Princeton Instruments, Trenton, NJ 08619 USA) at an excitation wavelength of white light and an exposure duration of 1 second.

The produced materials were initially assessed for their structure and morphology using XRD and SEM imaging techniques. As illustrated in Fig 2A , these materials reveal a nanosheet structure. The XRD pattern, displayed in Fig 2B , presents five unique peaks at positions (101), (012), (015), (110), and (113), suggestive of the MoS 2 -3R structure (PDF#17–0744, as analyzed with JADE software by MDI Materials Data). The resulting composite was also detected alongside MoS 2 -3R, (NH 4 ) 6 Mo 7 O 24 . However, the SEM image, shown in Fig 2A , emphasizes the existence of multilayer nanosheets within the hybrid material. This observation suggests that (NH 4 ) 6 Mo 7 O 24 plays a critical role in creating the lamellar MoS 2 , with NH 4 + ions occupying the layers in between and indicates that (NH 4 ) 6 Mo 7 O 24 either functionalizes the MoS 2 surface or decomposes into molecules. Furthermore, we explored the optical properties of the manufactured materials. An absorbance peak at 235 nm and a photoluminescence peak at 558 nm can be observed in Fig 2C .

Direct detection of B. subtilis DNA using fluorescent sensors based on the hybrid MoS 2 nanosheets

This study aims to design a simple optical sensing platform to detect a range of B. subtilis DNA concentrated from 23.6 to 130 aM, reflected by the number of copies of the testing sample from 16×106 to 16×107. In our experiment, the probe concentration was 30 nM (using 100 μL, which contains 1.83×1012 copies, a much more considerable amount than the number of ssDNA copies) and the concentration of sensing materials was varied. The sensors’ fluorescence was examined at hybrid MoS 2 concentrations of 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L, and 50 mg/L, and was presented in Fig 3.

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TIFF original image Download: Fig 3. PL spectra of sensors exposed to different concentrations of B. subtilis DNA. (A) PL spectra of sensors based on 30 mg/L MoS 2 while in contact with various B. subtilis DNA concentrations. When the DNA concentrations increased, the maximum intensities decreased. (B) The PL intensities of different sensors with multiple concentrations of hybrid MoS 2 nanosheets before in contact with B. subtilis DNA; The higher the sensing material concentration, the higher the PL. (C) The dependence of the intensities I at 558 nm of different sensors on the B. subtilis DNA concentrations. This relationship can be described by a linear function. (D) The ratios I/I 0 derived from the intensity at 558 nm of different sensors depending linearly on the DNA concentrations, where I 0 is the intensity of the sensor before contact with DNA. The error bars represent for standard deviations of nine measurements. https://doi.org/10.1371/journal.pone.0297581.g003

Fig 3A shows an example of PL spectra of sensors based on 30 mg/L hybrid MoS 2 nanosheets in contact with B. subtilis DNA. The other MoS 2 concentration sensors had similar shapes and quenching effects with DNA concentrations increased. They all had fluorescent peaks at 558 nm. The fluorescence spectra of all sensors based on 10 mg/L to 50 mg/L hybrid MoS 2 before adding DNA are presented in Fig 3B. Based on the intensity I and the ratio I/I 0 at the wavelength 558 nm, we established the calibration lines of I vs C and I/I 0 vs. C in Fig 3C & 3D, where I is the intensity of sensors with specific concentration of DNA; I 0 is the initial fluorescent intensity of sensors; C is the concentration of B. subtilis (aM). The operating functions were estimated and shown in Table 1. All sensors can operate linearly with high precision (all values of R2 were about 0.98). Among them, the sensors based on 50 mg/L have the highest sensitivity with the highest slope of -325. However, the 30 mg/L MoS 2 -based sensors have the best detection limit (LOD) of 18.7 aM and high sensitivity (slope of -233.8). In Table 1, the equation determined the limit of detection is LOD = blank signal + 3 standard derivations. Overall, the detection limits are slightly different, about 19 aM. That might be because the sensing material’s concentration is not much different (10 mg/L to 50 mg/L, only 40 mg/L difference).

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TIFF original image Download: Table 1. Linear relationship between intensity values at 558 nm and B. subtilis DNA concentrations. B. subtilis DNA concentrations changed from 23.6 aM– 130 aM of five sensors with concentrations of hybrid MoS 2 nanosheets of 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L. C is the concentration of B. subtilis DNA (aM). https://doi.org/10.1371/journal.pone.0297581.t001

In all experiments, the quenching effects have occurred and are reflected in the negative slopes of all operating functions. The quenching effect can be explained by the fact that in this study, we employed the NH 2 -5′-CCTACGGGAGGCAGCAGTAG-3′ probe to identify the complementary target B. subtilis DNA, adhering to the Watson-Crick base-pairing principles [8]. The probe was chemically modified with an amine group (NH 2 ) to enhance bonding to the MoS 2 surface. In the prepared nanomaterials, NH 4 + ions occupying the layers in between either functionalize the MoS 2 surface or help to bind with NH 2 - of the amine probe. Fig 4 provides a proposed schematic of the adsorbed single-stranded DNA (ssDNA) on the hybrid MoS 2 surface. The ssDNA can adhere to this surface, altering the dielectric properties of MoS 2 . However, when the ssDNA hybridizes with its complementary DNA, the resultant double-stranded DNA (dsDNA) establishes poor contact with the hybrid MoS 2 , distancing itself from the MoS 2 surface and changing the dielectric environment from DNA to water and reducing the photoluminescence [25]. The more complementary ssDNA added to the sensor, the more dsDNA was formed. Consequently, the photoluminescence was reduced more intensively. The intensity I at 558 nm, and the ratio of I/I 0 at 558 nm can be used to estimate the unknown B. subtilis DNA concentrations.

To validate the operational performance of this sensor, we prepared various DNA concentrations exceeding the limit of detection (LOD) and within the range defined by the calibration line. The concentrations examined were 74.3 aM, 97.5 aM, and 115.6 aM. The corresponding fluorescence measurements are depicted in Fig 5A. Utilizing the fluorescence values at 558 nm into the calibration line in Fig 5B, we determined the measured concentrations listed in Table 2. The calibrated concentrations were in good agreement with the experimental concentrations with small percentages of difference (smaller than 10%). When the test sample had a high concentration, the measured intensity was higher, and the precision improved. For example, with the sample of 115.6 aM, the difference between the actual concentration and the calibrated one was only 1.52%. The result confirms the reliability and repeatability of our proposed sensor.

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[1] Url: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0297581

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