Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual emission silver nanoclusters and catalytic hairpin amplification strategy

doi:10.21203/rs.3.rs-4697141/v1

Download PDF

Research Article

Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual emission silver nanoclusters and catalytic hairpin amplification strategy

https://doi.org/10.21203/rs.3.rs-4697141/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 16 Sep, 2024

Read the published version in Microchimica Acta →

You are reading this latest preprint version

Reliable, rapid and cost-efficient tools for the inspection and discrimination of genetically modified (GM) ingredients in food and food-related products are highly demanded to enforce relevant regulations in many countries. Herein, a dual-emission fluorescent biosensing method was developed for simultaneously quantitative analysis of CaMV35S and NOS in GM plants. Two designed hairpin DNA (H1, H2) sequences were used as templates to synthesize H1-AgNCs (λ_ex = 570 nm, λ_em = 625 nm) and H2-AgNCs (λ_ex = 470 nm, λ_em = 555 nm). By using H1-AgNCs and H2-AgNCs as dual-signal tags, combined with signal amplification strategy of magnetic separation to reduce background signal and an enzyme-free catalytic hairpin assembly (CHA) signal amplification strategy, a novel multi-target fluorescent biosensor was fabricated to detect multiple targets based on FRET between signal tags (donors) and magnetic Fe₃O₄ modified graphene oxide (Fe₃O₄@GO, acceptors). In the presence of the target NOS and CaMV35S, the hairpin structures of H1 and H2 can be opened respectively, and the exposed sequences will hybridize with the G-rich hairpin sequences HP1 and HP2 respectively, displacing the target sequences to participate in the next round of CHA cycle. Meanwhile, H1-HP1, H2-HP2 double-stranded DNA sequences (dsDNA) were formed, resulting the desorption of dsDNA from the surface of Fe₃O₄@GO due to weak π-π interaction between dsDNA and Fe₃O₄@GO, and leading to the fluorescence recovery of AgNCs. Under optimal conditions, the linear range of this fluorescence sensor were 5 ~ 300 nmol L^− 1 for NOS and 5 ~ 200 nmol L^− 1CaMV35S, and the LODs were 0.14 nmol L^− 1 and 0.18 nmol L^− 1, respectively. In addition, the fluorescence sensor has good selectivity for the detection of NOS and CaMV35S in GM soybean samples, showing the potential applications in GM screening.

Genetically modified crops

Biosensing

Fluorescence resonance energy transfer

Catalytic hairpin assembly

DNA template silver nanoclusters

In recent decades, the rapidly growing development of genetically modified (GM) crops has brought a huge potential for their application in agriculture. Many countries or regions and organizations have issued corresponding laws and regulations to ensure the healthy development of genetically modified technology and the legal application of GM crops [1, 2]. Therefore, there is an urgent requirement for the sensitive and selective detection of transgenic components in GM crops or foods [3, 4].

At present, the common methods for detection and identification of GM crops/foods are still those using PCR-based techniques [5, 6]. For example, real-time PCR (RT-PCR) with high sensitivity has long been considered as the golden method for quantitative detection of GM contents in GM crops/foods [7, 8]. However, the PCR-based methods still suffer from the requirement of dedicated and expensive equipment, highly professional personnel [2]. So far, different kinds of biosensors, including electrochemical biosensors [9, 10], lateral flow biosensors [11, 12], colorimetric biosensors [3], and fluorescence biosensors [2], have been constructed for the detection or screening GM crops/foods. Among these analytical methods, due to their advantages of high sensitivity and large throughput [13], fluorescence detection methods have been considered as potential methods for detection of GM contents in GM crops/foods. So far, various fluorescence biosensors including immunosensors and DNA sensors have been developed for detection of the promoter of the cauliflower mosaic virus 35s (CaMV35S) [14, 15], the terminator nopaline synthase (NOS) [14, 15], and biomarkers [16, 17] in GM crops.

Fluorescence resonance energy transfer (FRET) technique has been widely used in fluorescence detection of various targets [18–20]. The core of FRET-based detection system is fluorescent donor and acceptor. A variety of fluorescent donors including quantum dots [15], fluorescence dyes [18], metallic nanoclusters [19, 20], and so on, have been exploited for sensor fabrications. As one of the most common used metallic nanoclusters, silver nanoclusters (AgNCs) have been proved to be promising fluorescence labels in fabrication of fluorescence biosensors due to the in-situ synthesis on DNA templates [21, 22] and the advantages of long fluorescence lifetime, higher quantum yield, and better photostability [23]. They have been used as signal tags in FRET-based biosensors in detection of various targets [24, 25]. In terms of the fluorescent acceptors, a variety of nanomaterials [26] have been exploited as fluorescent acceptors. Graphene oxide (GO) is one of the materials that has been considered as an excellent fluorescent acceptor in FRET-based biosensing [27]. GO displays a strong capability to adsorb small molecules and single stranded DNA (ssDNA) to the surface through π-π stacking. Due to the unique property of GO, the fluorescence intensity of fluorescent donors can be quenched [28], displaying excellent quenching efficiency [16, 29]. Furthermore, the extraordinary quenching efficiency of GO can increase sensitivity while decreasing the background signal of the fluorescence.

In recent years, a few non-enzymatic nucleic acid amplification techniques have been widely used for DNA determination, for example, cascade amplification [30], entropy drive circuit (EDC) [31], hybrid chain reaction (HCR) [32] and catalytic hairpin amplification (CHA) [33]. In particular, CHA has generated great interest in the field of molecular sensing because it is a programmable DNA signal amplifier with the advantages of low cost, high sensitivity, and high efficiency that can stand on two partially complementary hairpins for target amplification [34]. On the basis of chain substitution reaction, CHA has adequate strength to perform signal amplification. As the signal increases “turn-on” fluorescence provides the analytical signal for target detection [35]. It is now being used widely for the identification of nucleic acids, small molecules, metal ions and proteins [33]. Traditional CHA-assisted fluorescence sensors often require complex fluorophore labeling. This not only increases the construction cost of the sensing platform but also the labeled fluorophores are susceptible to external factors. Therefore, the rational construction of label-free CHA-assisted biosensors is an urgent need, but also a great challenge.

In this study, the assembly of the fluorescent biosensor and its simultaneously detection strategy for two targets, CaMV35S and NOS, is shown in Scheme 1. Here, Fe₃O₄@GO was used as the fluorescence quenching material, the detection background was effectively reduced by two magnetic separation-steps [36], and the “turn-on” fluorescent biosensor based on magnetic separation was constructed in combination with the CHA signal amplification strategy. Firstly, Scheme 1A illustrates the CHA process with the detection of tDNA1 (NOS, shorted as T1) as an example, the DNA-recognition sensing procedure consists of T1 and two hairpin structures, H1 and HP1. T1 can hybridize with H1 to open the hairpin structure of H1, forming the H1-T1 duplex. When HP1 was added, it initiated a hybridization reaction with the 5’-end H1 (H1-T1) to form the H1-HP1 double stranded products and leading to the dissociation of the short single-stranded T1 simultaneously. Consequently, the released T1 sequence combined with free H1 to promote the next round of cycling reaction, forming numerous H1-HP1 duplexes. In Scheme 1B, the hairpin structures H1 and H2 were used as templates to synthesize the fluorescence donors: H1-AgNCs with red fluorescence (625 nm) and H2-AgNCs with green fluorescence (555 nm), respectively. After mixing with Fe₃O₄@GO, H1-AgNCs and H2-AgNCs were adsorbed onto the surface of Fe₃O₄@GO through a strong π-π stacking interaction. Due to the FRET interaction occurred between H1-AgNCs or H2-AgNCs and Fe₃O₄@GO, the fluorescence intensity of H1-AgNCs or H2-AgNCs was greatly quenched. The first magnetic separation was performed to remove excess H1-AgNCs and H2-AgNCs that were not adsorbed on the Fe₃O₄@GO surface, thereby reducing the background interference caused by non-quenching fluorescence. After adding the target T1, T2 and the G-rich sequences-hairpin structures of HP1 and HP2, the CHA cycle reactions were then initiated, and numerous H1-HP1 and H2-HP2 products were formed afterwards. Due to the weak binding ability of Fe₃O₄@GO to DNA duplex, H1-HP1 and H2-HP2 were desorbed from the surface of Fe₃O₄@GO. After the second magnetic separation, the fluorescence of H1-AgNCs and H2-AgNCs restored. Meanwhile, when the G-rich HP1 and HP2 were close to H1-AgNCs and H2-AgNCs, respectively, the fluorescence intensities were further enhanced. As the recovered intensities of H1-AgNCs and H2-AgNCs showed relevant to the concentrations of NOS and CaMV35S, the developed FRET-based biosensor can used to quantitative detection of targets.

Materials and instruments

All the details of materials and instruments were shown in Supporting Information.

Preparation of H1-AgNCs and H2-AgNCs

Here, H1 and H2 were used as DNA templates for preparing AgNCs with red and green fluorescence, respectively. H1 was used to prepare red color AgNCs. Ten µL of H1 (100 µmol/L) was mixed with 15 µL AgNO₃ (1.0 mmol/L) thoroughly, followed with the addition of fresh prepared NaBH₄ (15 µL, 100 µmol/L). The reduction reaction was carried out at 4 ℃ under continuously shaking for 12 h. The prepared nanomaterial named H1-AgNCs was stored at 4 ℃ for later use. The preparation of green color H2-AgNCs followed the same procedures as H1-AgNCs, except that the added volumes of AgNO₃ and NaBH₄ were 12 µL. By using rhodamine B [37] as the standard substance, the quantum yields of H1-AgNCs and H2-AgNCs were calculated to be 0.23 and 0.49, respectively.

Preparation of Fe₃O₄@GO

Fe₃O₄@GO was synthesized according to Hu’s method with modifications [18]. Briefly, GO (30 mg) was dispersed in ethylene glycol (5 mL) by ultrasonication for 2 h. The obtained solution was named solution A. A mixture named solution B was prepared by dissolving FeCl₃ (50 mg) and NaAC (100 mg) in ethylene glycol (5 mL). Then solution A and B were mixed with vigorously stirring for 1 h at room temperature. The resultant mixture was then transferred into an autoclave, and the reaction was conducted at 200 ℃ for 6 h. After cooling to room temperature, the precipitate was separated by magnetic force, and then repeatedly cleaned with water and ethanol. Finally, the obtained precipitate was dried in an oven and stored in a dryer for future use.

Preparation of real samples

The GM and non-GM soybeans were provided by the Technology Center of Hefei Customs District. The NOS and CaMV35S gene fragments in GM soybeans were extracted by using CTAB method [3]. First, GM soybeans (1.0 g) were crushed into powder, followed by the addition of Proteinase K (10 µL) and CTAB lysis buffer (1 mL). The mixture was heated at 65 ℃ for 1 h with intermittent vibration (time interval: 5 min). After that, the solution was cooled to room temperature and added with 700 µL of chloroform. The mixture was then kept shaking for 5 min, followed with centrifugation at 10,000 rpm for 10 min. Subsequently, the supernatant was collected and mixed with 400 µL of pre-chilled isopropanol. After incubating at -20 ℃ for 40 min, the solution was centrifuged (10,000 rpm) for 10 min at 4 ℃. The precipitate was collected and then washed with ethanol (75%, v/v) several times. The purified precipitate was air-dried, added with 50 µL of water, and stored at -20 ℃ for future use. The total DNA extract of non-GM soybeans was used as negative control, and its extraction steps were consistent with those described above.

PCR reactions were performed in a mixture containing 10 µL of 2× PCR buffer, 2 µL of the extracted DNA, 1 µL of forward and reverse primers (10 µmol/L), and 6 µL of ddH₂O. The amplification reaction was conducted under the conditions as follows: denatured at 94 ℃ for 5 min, followed by 30 cycles of amplification at 94 ℃ for 30 s, annealing at 57 ℃ for 30 s, extension at 72 ℃ for 30 s, and final extension at 72 ℃ for 4 min. The PCR products were verified by capillary electrophoresis, and the electrophoretic bands were observed with gel imaging analyzer.

Detection of the NOS and CaMV35S

Firstly, 37.5 µL of PBS buffer solution (0.1 mol L^− 1, pH 6.4) was added to the centrifuge tube, and 9.0 µL of Fe₃O₄@GO (2 mg mL^− 1) was added to the centrifuge tube and mixed with the buffer solution thoroughly. Then, 10 µL of H1-AgNCs and H2-AgNCs were added to the mixture, and the mixture was shaken for 10 min at room temperature and followed with a magnetic separation. After redispersion, 10 µL different concentration of CaMV35S and NOS targets (0.01, 0.1, 1, 5, 10, 30, 50, 100, 200, and 300 nmol L^− 1, respectively), and 6.75 µL of HP1 and HP2 (10 µmol L^− 1, respectively) were added. The supernatant was afterwards separated from solid particles with another magnetic separation step after incubation at 37 ℃ for 60 min.

Characterization of nanomaterials

The morphology and particle size of H1-AgNCs and H2-AgNCs were investigated by TEM, and the images and particle size distributions of the nanomaterials are shown in Fig. 1. The TEM images show that the prepared H1-AgNCs (Fig. 1A) and H2-AgNCs (Fig. 1C) were all in spherical shape and uniformly dispersed within the photographed range. According to the particle size histograms, the average particle sizes of H1-AgNCs (Fig. 1B) and H2-AgNCs (Fig. 1D) were about 2.83 nm and 2.91 nm, respectively. The morphology GO and Fe₃O₄@GO was characterized by SEM, and the images are shown in Fig. 1E and D, respectively. As shown in Fig. 1E, GO had a layered structure, and several folds could be clearly observed. After surface modification with Fe₃O₄ through chemical reduction, the surface of GO was uniformly dispersed by Fe₃O₄ nanoparticles. The average particle size of immobilized Fe₃O₄ was estimated to be 200 nm.

<Fig. 1>

The elemental compositions of H1-AgNCs, H2-AgNCs, GO and Fe₃O₄@GO were characterized by XPS, and the spectra are shown in Fig. 2. It can be seen from Fig. 2A and C that the O1s, N1s, C1s, and P2p peaks of H1-AgNCs and H2-AGNCs can be observed clearly at the binding energy of about 532.4 eV, 399.6 eV, 284.8 eV and 132.4 eV. In addition, the Ag3d spectra in H1-AgNCs (Fig. 2B) and H2-AgNCs (Fig. 2D) show two characteristic peaks located at approximately 373.8 eV and 368.1 eV, which can be attributed to Ag3d_3/2 and Ag3d_5/2 [20], respectively. The emerging peaks of P2p and Ag3d demonstrates the successful preparation of bi-fluorescence AgNCs using harpin sequences as templates. Figure 2E and F are the XPS survey spectra of GO and Fe₃O₄@GO. As shown in Fig. 2E, the peaks at 532.6 eV and 286.8 eV were assigned to O1s and C1s, respectively. The molar ratio of C to O in GO were calculated to be about 2.05. In addition to the observed peaks of O1s and C1s, the peak emerged at 711.3 eV assigned to Fe2p can be observed in Fe₃O₄@GO (Fig. 2F), demonstrating the success of surface modification of GO. Furthermore, the molar ratio of C to O in Fe₃O₄@GO were calculated to be about 5.6, which is much larger than that in GO. This indicates that during hydrothermal treatment, GO was partially deoxidized by removing most of the epoxy and hydroxyl functional groups contained in GO, and chemical reduced graphene oxide (rGO) was formed.

<Fig. 2>

Sensing principle and feasibility of the fluorescence biosensor

The NOS/CaMV35S detection principle is illustrates in Scheme 1. The scheme shows that in the present fluorescence sensing system, the fluorescence intensity of H1-AgNCs/H2-AgNCs was firstly quenched by Fe₃O₄@GO due to the FRET interaction between H1/H2 and Fe₃O₄@GO. After addition of target NOS/CaMV35S and with the assistance of CHA strategy, the fluorescence intensity of H1-AgNCs/H2-AgNCs can be greatly recovered. The feasibility and sensing strategy of dual-signal fluorescent biosensor were verified by the detection of NOS (100 nmol L^− 1) and CaMV35S (100 nmol L^− 1), and the obtained fluorescence spectra are shown Fig. 3A and B. As shown in Fig. 3A, the fluorescent intensity of H1-AgNCs was dramatically quenched by the addition of Fe₃O₄@GO. After conducting magnetic separation and redispersing in PBS, the intensity of H1-AgNCs (Fig. 3A, curve b) was measured and calculated to be 14.5% of the initial intensity of H1-AgNCs (Fig. 3A, curve a). Then, with the addition of target NOS and performing magnetic separation, the intensity of H1-AgNCs (Fig. 3A, curve c) was recovered to 18.0% of the initial intensity of H1-AgNCs. However, the intensity could be greatly enhanced by the addition target NOS and HP1 into the sensing system, and the recovered intensity reached 50.9% of the initial intensity of H1-AgNCs (Fig. 3A, curve d). Similar trends can be observed in verifying the feasibility of target CaMV35S detection (Fig. 3B). The intensity of redispersed H2-AgNCs (Fig. 3B, curve b) maintained 19.9% of the initial intensity of H2-AgNCs (Fig. 3B, curve a), 28.6% of the initial after addition of CaMV35S (Fig. 3B, curve c), and 54.7% of the initial after the addition of CaMV35S and HP2 (Fig. 3B, curve d). The hairpin-structured H1/H2 and Fe₃O₄@GO have strong π-π interaction. With the addition of targets (NOS and CaMV35S) that can open H1 and H2, partially double stranded DNA sequences formed. However, this sequence couldn’t fully release from the surface of Fe₃O₄@GO due to relatively high electric interaction between the sequence and GO. As a result, without CHA strategy, the fluorescence intensities of H1-AgNCs and H2-AgNCs were only slightly recovered. When using the CHA strategy, which involved the simultaneous presence of the targets (NOS and CaMV35S) and hairpins (HP1 and HP2), the fluorescence recoveries of H1-AgNCs and H2-AgNCs were greatly improved. When applying CHA strategy, the added HP1 and HP2 can hybridize with the H1 and HP2, respectively, to form double stranded complexes HP1-H1-AgNCs and HP2-H2-AgNCs, while releasing NOS and CaMV35S that bound to H1 and H2. The released NOS and CaMV35S will enter the next cycle to open H1 and H2, respectively. Due to the weaker affinity between the double stranded complexes and GO compared to the hairpin structure, HP1-H1-AgNCs and HP2-H2-AgNCs detached from the surface of GO, thereby restoring the fluorescence intensity of H1-AgNCs and H2-AgNCs dramatically. These results indicate that the detection signal can be significantly enhanced only when the targets, HP1 and HP2 coexist in the detection system.

<Fig. 3>

Analytical performance of biosensors

Before fluorescence detection of NOS and CaMV35S, several parameters were optimized to achieve the optimal analytical performance of the biosensor. Firstly, the parameters for the preparation of H1-AgNCs and H2-AgNCs were optimized to obtain AgNCs with high fluorescent intensity. These parameters included the molar ratio of hairpin template (H1 and H2) to Ag⁺, pH value and reaction time. Fig. S1 A-C displays the optimization results of the preparation conditions for H1-AgNCs. As shown in these figures, we can conclude that the prepared H1-AgNCs can reach the highest fluorescence intensity under the following preparation conditions: the molar ratio of H1 to Ag⁺ was 1:15, the pH value was 6.4, and the reaction time was 60 min. In terms of optimal conditions for the preparation of H2-AgNCs, the molar ratio of H2 to Ag⁺, pH value and reaction time were 1:12, 6.6 and 60 min, respectively. Secondly, the added volume of Fe₃O₄@GO (2 mg mL^− 1) was also optimized (Fig. S2). As shown in the figure, the fluorescence intensity of H1-AgNCs (Fig. S2A) and H2-AgNCs (Fig. S2B) decreased gradually with the increased volume of Fe₃O₄@GO suspension. When the added volume of Fe₃O₄@GO was reached 9 µL, the fluorescence intensity of H1-AgNCs and H2-AgNCs maintained 14.5% and 19.9% of the initial intensity of H1-AgNCs and H2-AgNCs, respectively. When the added volume of Fe₃O₄@GO increased to 12 µL, the fluorescence intensity of H1-AgNCs and H2-AgNCs further decreased, maintaining 8.4% and 8.6% of their initial intensity, respectively. However, after recovering with 100 nmol L^− 1 CaMV35S and conducting CHA strategy, the calculated (F˗F₀)/F₀ (F refers to fluorescence intensity of the recovered intensity, F₀ refers to the fluorescence intensity of H2-AgNCs-Fe₃O₄@GO redispersed in PBS) was only 0.81; whereas when quenched with 9 µL Fe₃O₄@GO, the value of (F˗F₀)/F₀ was 1.77. This demonstrates that after quenching with 9 µL Fe₃O₄@GO, target CaMV35S and CHA strategy can effectively recover the fluorescence intensity of H2-AgNCs. Similar results can be obtained when using 50 and 200 nmol L^− 1 CaMV35S to restore the fluorescence intensity of H2-AgNCs. Therefore, 9 µL was chosen as the added volume of Fe₃O₄@GO (2 mg mL^− 1) in this experiment. Finally, the parameters for the detection of NOS and CaMV35S were optimized to achieve the best analytical performance of the biosensor. These parameters included the concentration of HP1/HP2, hybridization temperature and time. As shown in Fig. S3, the optimal experimental conditions for detection of NOS and CaMV35S should be carried out under the following conditions: (1) The concentrations of HP1 and HP2 were both 675 nmol L^− 1 (Fig. S3A); (2) The hybridization temperature was 37 ℃ (Fig. S3B); (3) The hybridization time was 60 min (Fig. S3C).

Under the optimal experimental conditions, the fluorescence biosensor was used to detect NOS and CaMV35S, and the responses to various concentrations of tDNAs are displayed in Fig. 4A and C. As shown in the figure, due to the CHA reaction and the increase of NOS and CaMV35S concentration, the recovered fluorescence intensity of H1-AgNCs (Fig. 4A) and H2-AgNCs (Fig. 4C) increased expectedly. By analyzing the fluorescence intensity of H1-AgNCs (I₁) for NOS and H2-AgNCs (I₂) for CaMV35S with the concentrations of NOS (C_tDNA1) and CaMV35S (C_tDNA2), two good linear relationships were obtained. As displayed in Fig. 4B, I₁ was linear with the common logarithms of NOS concentration in range of 5 ~ 300 nmol L^− 1, and the fitted line equation was written as I₁ = 328.21lg(C_tDNA1/ nmol L^− 1) + 125.26 (R² = 0.996). The limit of detection (LOD) was calculated to be 0.14 nmol L^− 1 (S/N = 3). The calibration plot (Fig. 4C) showed linear relationship between I₂ and the common logarithms of CaMV35S concentration in range of 5 ~ 200 nmol L^− 1. The equation was presented as I₂ = 635.24lg(C_tDNA2/ nmol L^− 1) + 322.09 (R² = 0.994). The LOD was calculated to be 0.18 nmol L^− 1 (S/N = 3).

<Fig. 4.>

The analytical performances in linear range and LOD of our fluorescence biosensor were compared with other reported fluorescence biosensors [14, 29, 38–40], and the results are listed in Table 1. As can be seen from the table, our biosensor also showed good analytical performance in terms of linear range and detection limit.

Table 1

Comparison of different fluorescent biosensors for detecting GMO
Method and material	Target	Linear range (nmol L^− 1)	LOD (nmol L^− 1)	Ref.
Fluorescence biosensor, incorporation of bicolor CdTe QDs carried by silica nanospheres	NOS	0.2 ~ 800	0.07	[14]
	CaMV35S	0.1 ~ 800	0.04	[14]
FRET based biosensor, bicolor CdTe QDs and MWCNTs@GONRs	NOS	1.5 ~ 1000	0.5	[15]
FRET based biosensor, bicolor CdTe QDs and MWCNTs@GONRs	CaMV35S	1.2 ~ 900	0.35	[15]
FRET based biosensor, CDs and GO	NOS	0.5 ~ 50	0.008	[29]
Ratio fluorescence biosensor, dual-emission AgNCs	CaMV35S	10 ~ 1000	1.5	[38]
FRET based biosensor, FAM-ssDNA and Fe-MIL-88	CaMV35S	5×10^− 3 ~ 50	1.84×10^− 4	[41]
FRET based biosensor, bicolor AgNCs and Fe₃O₄@GO	NOS	5 ~ 300	0.14	This work
FRET based biosensor, bicolor AgNCs and Fe₃O₄@GO	CaMV35S	5 ~ 200	0.18	This work
Note: CDs: carbon dots; MWCNTs@GONRs: multiwalled carbon nanotubes@graphene oxide nanoribbons; FAM-ssDNA: single-strand DNA-labeled FAM dye.

<Table 1.>

The analytical specificity of the biosensor was further investigated by detecting different mismatched DNA sequences. Here, completely non complementary sequence (NC, 500 nmol L^− 1), single base mismatch DNA sequences (Mis1-1 and Mis1-2, 500 nmol L^− 1), two base mismatch DNA sequences (Mis2-1 and Mis2-2, 500 nmol L^− 1) and three base mismatch DNA sequences (Mis3-1 and Mis3-2, 500 nmol L^− 1) were introduced into the detecting system in place of the target NOS and CaMV35S (100 nmol L^− 1). In addition, the biosensor was also used to detect 100 nmol L^− 1 NOS and CaMV35S in a mixture containing the above-mentioned DNA sequences. Figure 5A and B show the recorded fluorescence intensity for the detection of NOS and CaMV35S in buffer solution, mismatched sequences and NOS/CaMV35S in a mixture containing various mismatched sequences. The results revealed that the biosensor can only give strong fluorescence response when detecting NOS (Fig. 5A) and CaMV35S (Fig. 5B), demonstrating good selectivity of the biosensor.

We also investigated the stability of the biosensor by evaluating the storage stability of H1-AgNCs and H2-AgNCs, and the results are displayed in Fig. 5C and D. After 5 days storage at 4 ℃, the fluorescence intensity of H1-AgNCs and H2-AgNCs decreased by 2.80% and 1.98% compared to the initial intensity, respectively. The intensity of H1-AgNCs and H2-AgNCs further decreased along with the increasing storage time, and their intensity decreased by 14.25% and 15.64% respectively after 20 days of storage. Therefore, the prepared H1-AgNCs and H2-AgNCs maintained stable fluorescence intensity within 10 days, which can ensure the accuracy and stability of our fabricated biosensor.

<Fig. 5.>

Detection of NOS and CaMV35S segments in real samples

The biosensor was finally applied for the detection of NOS and CaMV35S segments in the extracted DNA from GM soybeans. PCR products using the extracted DNA as template were verified by capillary electrophoresis (Fig. 6A and Fig. S4). The NOS and CaMV35S segments in PCR product of soybeans appeared at 250 (lane A03 and A05) 410 bp (lane A02 and A04), respectively. No such bands were found at this position in non-GM soybeans (lane A08 and A09) and GM soybeans without primers (lane A06 and A07). As displayed in Fig. 6B and C, the biosensor gave strong response to the PCR product of GM soybeans, while almost no response was detected to the PCR products of non-GM soybean and GM soybeans without primers. According to the fitted line equations, the concentrations of NOS and CaMV35S segments in GM soybean PCR product of were calculated to be 28.25 ± 0.47 nmol/L and 15.96 ± 0.35 nmol/L, respectively. The reliability and accuracy of the biosensor in detecting target segments in real sample was verified using standard addition method, and the results are listed in Table S2. As indicated in the table, the recoveries were in the range of 88.22 ~ 106.12%. The results indicate that the proposed biosensor has good reliability and accuracy in detecting targets in real samples.

<Fig. 6.>

In conclusion, we developed a FRET-based fluorescent biosensor for the detection of NOS and CaMV35S of GM plants. The dual target monitoring biosensor exploited the FRET effect between bi-fluorescence AgNCs and Fe₃O₄@GO combining with CHA strategy. The biosensing method was used to detect NOS and CaMV35S segments in real GM soybeans simultaneously, suggesting a potential method for detecting specific DNA sequences in GMO.

Author contribution Yongkang Ye: Methodology, Formal analysis, Funding acquisition, Writing – original draft, Writing – review & editing. Yinghui Zhai: Experiments, Formal analysis, Writing – original draft, Validation. Chenlu Zhang: Experiments. Shaopeng Wang: Experiments. Yuexi Lu: Experiments, Funding acquisition. Xiaodong Cao: Methodology, Writing – original draft, Writing – review & editing, Project administration, Funding acquisition. Shudong He: Resources, Instruments and Equipment for experiments. Haisong Zheng: Resources, Instruments and Equipment for experiments. Yunfei Li: Resources, Instruments and Equipment for experiments, Funding acquisition. Yunlai Tao: Resources, Funding acquisition. All authors who have made substantial contributions to the work are listed in the manuscript.

Funding This work was supported by the Key Project of Ningxia Natural Science Foundation (No. 2022AAC02046), the Anhui Province Science and Technology Major Special Project (No. 202103a07020003), the Key Research and Development Project of Anhui Province (No. 202104a06020005), the National College Students Innovative Entrepreneurial Training Program (No. 202310359112).

Data availability The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Alarcon CM, Shan G, Layton DT, Bell TA, Whipkey S, Shillito RD (2019) Application of DNA- and Protein-Based Detection Methods in Agricultural Biotechnology. J Agric Food Chem 67(4): 1019–1028. https://doi.org/10.1021/acs.jafc.8b05157.
Liu Y, Zhou S, Sun H, Dong J, Deng L, Qi N, Wang Y, Huo D, Hou C (2022) Ultrasensitive fluorescent biosensor for detecting CaMV 35S promoter with proximit y extension mediated multiple cascade strand displacement amplification and CRISPR/Cpf 1. Anal Chim Acta 1215: 339973, https://doi.org/10.1016/j.aca.2022.339973.
Cao X, Xia Z, Yan W, He S, Xu X, Wei Z, Ye Y, Zheng H (2020) Colorimetric biosensing of nopaline synthase terminator using Fe₃O₄@Au and hemin-functionalized reduced graphene oxide. Anal Biochem 602: 113798. https://doi.org/10.1016/j.ab.2020.113798.
Li J, Zhai S, Gao H, Xiao F, Li Y, Wu G, Wu Y (2021) Development and assessment of a duplex droplet digital PCR method for quantification of GM rice Kemingdao. Anal Bioanal Chem 413: 4341–4351. https://doi.org/10.1007/s00216-021-03390-9.
Park JE, Lee DG, Kim HR, Kim MJ, Ki HY m, Kim HJ (2022) Development of ultrafast PCR assays for the event-specific detection of eleven approved genetically modified canola events in South Korea. Food Chem 373: 131419. https://doi.org/10.1016/j.foodchem.2021.131419.
Niu C, Xu Y, Zhang C, Zhu P, Huang K, Luo Y, Xu W (2018) Ultrasensitive single fluorescence-labeled probe-mediated single universal primer-multiplex-droplet digital polymerase chain reaction for high-throughput genetically modified organism screening. Anal Chem 90(9): 5586–5593. https://doi.org/10.1021/acs.analchem.7b03974.
Weidner C, Edelmann S, Moor D, Lieske K, Savini C, Jacchia S, Sacco MG, Mazzara M, Lämke J, Eckermann KN, Emons H, Mankertz J, Grohmann L (2022) Assessment of the real-time PCR method claiming to be specific for detection and quantification of the first commercialised genome-edited plant, Food Anal Methods 15: 2107–2125. https://doi.org/10.1007/s12161-022-02237-y.
Cottenet G, Blancpain C, Sonnard V, Chuah PF (2019) Two FAST multiplex real-time PCR reactions to assess the presence of genetically modified organisms in food. Food Chem 274: 760–765. https://doi.org/10.1016/j.foodchem.2018.09.050.
Ye Y, Mao S, He S, Xu X, Cao X, Wei Z, Gunasekaran S (2020) Ultrasensitive electrochemical genosensor for detection of CaMV35S gene with Fe₃O₄-Au@Ag nanoprobe. Talanta 206: 120205. https://doi.org/10.1016/j.talanta.2019.120205.
Chen D, Zhang M, Ma M, Hai H, Li J, Shan Y (2019) A novel electrochemical DNA biosensor for transgenic soybean detection based on triple signal amplification. Anal Chim Acta 1078: 24–31. https://doi.org/10.1016/j.aca.2019.05.074.
Wang X, Chen Y, Chen X, Peng C, Wang L, Xu X, Wu J, Wei W, Xu J (2020) A highly integrated system with rapid DNA extraction, recombinase polymerase amplification, and lateral flow biosensor for on-site detection of genetically modified crops. Anal Chim Acta 1109: 158–168. https://doi.org/10.1016/j.aca.2020.02.044.
Li S, Tian J, Zhu L, Huang K, Chu H, Xu W (2022) Functional nucleic acid lateral flow magnetic biosensor based on blocking the super PCR and magnetic test strip for rapid detection of genetically modified maize MON810. Anal Chim Acta 1202: 339660. https://doi.org/10.1016/j.aca.2022.339660.
Schaaf TM, Kleinboehl E, Yuen SL, Roelike LN, Svensson B, Thompson AR, Cornea RL, Thomas DD (2020) Live-cell cardiac-specific high-throughput screening platform for drug-like molecules that enhance Ca²⁺ transport. Cells 9: 1170. https://doi.org/10.3390/cells9051170.
Li Y, Hao N, Luo S, Liu Q, Sun L, Qian J, Cai J, Wang K (2020) Simultaneous detection of TNOS and P35S in transgenic soybean based on magnetic bicolor fluorescent probes. Talanta 212: 120764. https://doi.org/10.1016/j.talanta.2020.120764.
Li Y, Sun L, Qian J, Long L, Li H, Liu Q, Cai J, Wang K (2017) Fluorescent “on-off-on” switching sensor based on CdTe quantum dots coupled with multiwalled carbon nanotubes@graphene oxide nanoribbons for simultaneous monitoring of dual foreign DNAs in transgenic soybean. Biosens Bioelectron 92: 26–32. https://doi.org/10.1016/j.bios.2017.01.057.
Yin K, Liu A, Shangguan L, Mi L, Liu X, Liu Y, Zhao Y, Li Y, Wei W, Zhang Y, Liu S (2017) Construction of iron-polymer-graphene nanocomposites with low nonspecific adsorption and strong quenching ability for competitive immunofluorescent detection of biomarkers in GM crops. Biosens Bioelectron 90: 321–328. https://doi.org/10.1016/j.bios.2016.11.070.
Kanagasubbulakshmi S, Kadirvelu K (2021) Paper-based simplified visual detection of Cry2Ab insecticide from transgenic cottonseed samples using integrated quantum dots-IgY antibodies. J Agric Food Chem 69: 4074–4080. https://doi.org/10.1021/acs.jafc.0c07180.
Hu LY, Niu CG, Wang XY, Huang DW, Zhang L, Zeng GM (2017) Magnetic separate “turn-on” fluorescent biosensor for Bisphenol A based on magnetic oxidation graphene. Talanta 168: 196–202. https://doi.org/10.1016/j.talanta.2017.03.055.
Guo Y, Zhang Y, Pei R, Cheng Y, Xie Y, Yu H, Yao W, Li HW, Qian H (2019) Detecting the adulteration of antihypertensive health food using G-insertion enhanced fluorescent DNA-AgNCs. Sensors Actuators B Chem 281: 493–498. https://doi.org/10.1016/j.snb.2018.10.101.
Ye Y, Liu Y, He S, Xu X, Cao X, Ye Y, Zheng H (2018) Ultrasensitive electrochemical DNA sensor for virulence invA gene of Salmonella using silver nanoclusters as signal probe. Sensors Actuators B Chem 272: 53–59. https://doi.org/10.1016/j.snb.2018.05.133.
Xiao Y, Shu F, Wong KY, Liu Z (2013) Förster resonance energy transfer-based biosensing platform with ultrasmall silver nanoclusters as energy acceptors. Anal Chem 85(18): 8493–8497. https://doi.org/10.1021/ac402125g.
Yang CL, Zhang YQ, He JY, Li MD, Yuan R, Xu WJ (2022) Target deoxyribonucleic acid-recycled lighting-up amplifiable ratiometric fluorescence biosensing of bicolor silver nanoclusters hosted in a switchable deoxyribonucleic acid construct. Anal Chem 94(18): 6703–6710. https://doi.org/10.1021/acs.analchem.1c05445.
Yang M, Chen X, Su Y, Liu H, Zhang H, Li X, Xu W (2020) The fluorescent palette of DNA-templated silver nanoclusters for biological applications. Front Chem 8: 601621. https://doi.org/10.3389/fchem.2020.601621.
He JY, Shang X, Yang CL, Zuo SY, Yuan R, Xu WJ (2021)Antibody-responsive ratiometric fluorescence biosensing of biemissive silver nanoclusters wrapped in switchable DNA tweezers. Anal Chem 93 (33): 11634–11640. https://doi.org/10.1021/acs.analchem.1c02444.
Liu F, Bing T, Shangguan D, Zhao M, Shao N (2016) Ratiometric fluorescent biosensing of hydrogen peroxide and hydroxyl radical in living cells with lysozyme-silver nanoclusters: lysozyme as stabilizing ligand and fluorescence signal unit. Anal Chem 88 (21): 10631–10638. https://doi.org/10.1021/acs.analchem.6b02995.
Wu L, Huang C, Emery BP, Sedgwick AC, Bull SD, He XP, Tian H, Yoon J, Sessler JL, James TD (2020) Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents. Chem Soc Rev 49: 5110–5139. https://doi.org/10.1039/c9cs00318e.
Tian F, Lyu J, Shi J, Yang M (2017) Graphene and graphene-like two-denominational materials based fluorescence resonance energy transfer (FRET) assays for biological applications. Biosens Bioelectron 89: 123–135. https://doi.org/10.1016/j.bios.2016.06.046.
He Y, Cheng Y, Wen X (2022) A design of red emission CDs-based aptasensor for sensitive detection of insulin via fluorescence resonance energy transfer. Spectrochim Acta - Part A Mol Biomol Spectrosc 280: 121497. https://doi.org/10.1016/j.saa.2022.121497.
He Y, Fan Z (2018) A novel biosensor based on DNA hybridization for ultrasensitive detection of NOS terminator gene sequences. Sensors Actuators B Chem 257: 538–544. https://doi.org/10.1016/j.snb.2017.10.183.
Yuan Y, Ma Y, Luo L, Wang Q, Huang J, Liu J, Yang X, Wang K (2018) Ratiometric determination of human papillomavirus-16 DNA by using fluorescent DNA-templated silver nanoclusters and hairpin-blocked DNAzyme-assisted cascade amplification. Microchim Acta 186: 613. https://doi.org/10.1007/s00604-019-3732-y.
Chen X, Yang L, Liang S, Dang P, Jin D, Cheng Z, Lin J (2021) Entropy-driven strand displacement reaction for ultrasensitive detection of circulating tumor DNA based on upconversion and Fe₃O₄ nanocrystals. Sci China Mater 64: 2593–2600. https://doi.org/10.1007/s40843-020-1649-1.
Zhang S, Wang K, Li KB, Shi W, Jia WP, Chen X, Sun T, Han DM (2017) A DNA-stabilized silver nanoclusters/graphene oxide-based platform for the sensitive detection of DNA through hybridization chain reaction. Biosens Bioelectron 91: 374–379. https://doi.org/10.1016/j.bios.2016.12.060.
Xiong Y, Dai J, Zhang Y, Zhou C, Yuan H, Xiao D (2021) A label-free fluorescent biosensor based on a catalyzed hairpin assembly for HIV DNA and lead detection. Anal Methods 13: 2391–2395. https://doi.org/10.1039/d1ay00410g.
Zou R, Wang S, Chen C, Chen X, Gong H, Cai C (2021) An enzyme-free DNA circuit-assisted MoS₂ nanosheet enhanced fluorescence assay for label-free DNA detection. Talanta. 222: 121505. https://doi.org/10.1016/j.talanta.2020.121505.
Tao F, Fang J, Guo Y, Tao Y, Han X, Hu Y, Wang J, Li L, Jian Y, Xie G (2018) A target-triggered biosensing platform for detection of HBV DNA based on DNA walker and CHA. Anal Biochem 554: 16–22. https://doi.org/10.1016/j.ab.2018.05.024.
Cao X, Zhang K, Yan W, Xia Z, He S, Xu X, Ye Y, Wei Z, Liu S (2020) Calcium ion assisted fluorescence determination of microRNA-167 using carbon dots–labeled probe DNA and polydopamine-coated Fe₃O₄ nanoparticles. Microchim Acta 187: 212. https://doi.org/10.1007/s00604-020-4209-8.
Guo Y, Zhang Y, Pei R, Cheng Y, Xie Y, Yu H (2019) Detecting the adulteration of antihypertensive health food using G-insertion enhanced fluorescent DNA-AgNCs. Sensors Actuators B Chem 281: 493–498. https://doi.org/10.1016/j.snb.2018.10.101.
Alipour M, Jalili S, Shirzad H, Dezfouli EA, Fouani MH, Sadeghan AA, Bardania H, Hosseinkhani S (2020) Development of dual-emission cluster of Ag atoms for genetically modified organisms detection. Microchim Acta 187: 628. https://doi.org/10.1007/s00604-020-04591-2.
Qiu B, Zhang YS, Lin YB, Lu YJ, Lin ZY, Wong KY, Chen GN (2013) A novel fluorescent biosensor for detection of target DNA fragment from the transgene cauliflower mosaic virus 35S promoter. Biosens Bioelectron 41: 168–171. https://doi.org/10.1016/j.bios.2012.08.017.
Wu L, Deng J, Tan X, Yin W, Ding F, Han H (2018) Ratiometric fluorescence sensor for the sensitive detection of Bacillus thuringiensis transgenic sequence based on silica coated supermagnetic nanoparticles and quantum dots. Sensors Actuators B Chem 254: 206–213. https://doi.org/10.1016/j.snb.2017.07.021.
Liu Y, Zhou S, Dong J, Sun H, Deng L, Ma Y, Zhao D, Huo D, Hou C (2023) Rapid and ultrasensitive fluorescence sensing platform based on nanometer-sized metal-organic frameworks for transgenic CaMV 35S promoter detection. ACS Appl Nano Mater 6(8): 7022–7030. https://doi.org/10.1021/acsanm.3c01091.

No competing interests reported.

SupportingInformation0709.docx

Download PDF

Journal Publication

published 16 Sep, 2024

Read the published version in Microchimica Acta →

Reviews received at journal
12 Aug, 2024
Reviewers agreed at journal
11 Aug, 2024
Reviewers agreed at journal
08 Aug, 2024
Reviews received at journal
31 Jul, 2024
Reviewers agreed at journal
19 Jul, 2024
Reviewers invited by journal
13 Jul, 2024
Editor assigned by journal
10 Jul, 2024
Submission checks completed at journal
10 Jul, 2024
First submitted to journal
06 Jul, 2024

You are reading this latest preprint version

Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual emission silver nanoclusters and catalytic hairpin amplification strategy

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Experimental

Materials and instruments

Preparation of H1-AgNCs and H2-AgNCs

Preparation of Fe₃O₄@GO

Preparation of real samples

Detection of the NOS and CaMV35S

Results and discussion

Characterization of nanomaterials

<Fig. 1>

<Fig. 2>

Sensing principle and feasibility of the fluorescence biosensor

<Fig. 3>

Analytical performance of biosensors

<Fig. 4.>

<Table 1.>

<Fig. 5.>

Detection of NOS and CaMV35S segments in real samples

<Fig. 6.>

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual emission silver nanoclusters and catalytic hairpin amplification strategy

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Experimental

Materials and instruments

Preparation of H1-AgNCs and H2-AgNCs

Preparation of Fe3O4@GO

Preparation of real samples

Detection of the NOS and CaMV35S

Results and discussion

Characterization of nanomaterials

<Fig. 1>

<Fig. 2>

Sensing principle and feasibility of the fluorescence biosensor

<Fig. 3>

Analytical performance of biosensors

<Fig. 4.>

<Table 1.>

<Fig. 5.>

Detection of NOS and CaMV35S segments in real samples

<Fig. 6.>

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Preparation of Fe₃O₄@GO