Microarray-based assay design for visual identification of pathogenic genes

A microarray-based detection method using terminal nucleotide addition reaction for visual identification of pathogenic genes was developed through the fabrication of an unmodified COC-based microarray chip and optimization of colorimetric signal formation. As shown in Fig. 1, the capture probe DNA capable of partially complementarily binding to the target gene was consistently arranged by a contact microarray spotter and fixed on the surface of the COC chip. The target gene loaded onto the microarray chip was hybridized with the capture probe DNA that had sequence complementarity and b-dUTP was continuously added to the 3ʹ end with the TdT reaction. The Poly b-dUTP synthesized selectively forms a complex with s-AuNPs through a biotin-streptavidin interaction, causing the formation of black spots that can be observed with the naked eye through an additional silver enhancement reaction for 4 min. However, in the case the target gene was absent, the synthesis of poly b-dUTP in the array spot was inhibited because TdT could not use a phosphate-protected capture probe at the 3ʹ as a reaction substrate. This microarray-based detection method saves time, cost, and labor required for multi-target or multiple samples analysis as it can analyze numerous targets simultaneously compared to the general real-time polymerase chain reaction that uses fluorescent probes within the range where their absorption wavelengths do not overlap each other. In addition, since the colorimetric signal formed in the microarray spot minimizes the use of complex and expensive equipment for signal analysis, intuitive result analysis is possible, and is cost-effective compared to the fluorescence signal-based microarray detection method. In the past, for the detection of a target gene, a sandwich assay using simple and direct binding between target DNA and AuNP probes was usually performed on a colorimetric microarray chip [25, 26]. On the other hand, in our method, the generation of the b-dUTP tail from one target gene by using the TdT reaction has the advantage of binding a larger amount of AuNPs to the target gene. These characteristics act as factors that amplify the colorimetric signal.

Fig. 1
figure 1

Schematic illustration of the microarray-based detection method for visually identifying pathogenic genes

Optimization of spotting conditions for DNA microarray chip fabrication

To ensure uniform microarray spot formation on the non-modified COC chip surface, it was sequentially washed with water, ethanol, and isopropanol before use and treated with oxygen plasma to induce hydrophilicity of the COC surface. To produce high-quality DNA array spots using a contact microarray system, the pickup height and speed of the microarray pins were determined to be 1.5 mm and 3 s, respectively (data not shown). And to prevent cross-contamination between samples, 5 cycles of DW washing for 30 s and air drying for 30 s were determined as the optimal washing conditions (data not shown). Optimal spotting buffer conditions were also investigated, as shown in Fig. 2. Two types of spotting solutions were compared. For buffer A (150 mM sodium phosphate buffer with 0.01% tween 20) [19], the fluorescent probe DNA was intensively fixed at the edge of the spot, while commercial buffer B (1× micro spotting solution plus produced by the Arrayit Corporation) tended to be non-uniformly fixed at the center of the spot. Because the uniform distribution of the capture probe DNAs on the microarray spot was essential for the efficiency of subsequent hybridization and enzymatic reactions, we further tested the mixing conditions of buffers A and B. We found that uniformly fixed spots formed when the two buffers were mixed at a volume ratio of 1:1 (Fig. 2C). This phenomenon may be influenced by the concentration of surfactant present in the spotting buffer. In previous studies, it has been reported that when the amount of surfactant in the spotting buffer is small, the fluorescence signal of the microarray is concentrated in the center of the spot, and when the amount is large, a coffee ring pattern occurs [19, 27]. However, in determining the quality of microarray spots, not only the concentration of the surfactant but also other factors such as the surface properties of the solid substrate, the viscosity of the solution, drying conditions, and microarray type were thought to act in a complex way.

Fig. 2
figure 2

Fluorescence scan image of fluorescent probe DNA immobilized on a COC chip to determine the optimal spotting buffer conditions. The scale bar is 500 μm

Optimization of experimental parameters

To optimize the TdT reaction conditions, the optimal reaction time and concentration of b-dUTP were determined. Regarding the TdT reaction time, the mean grey values of spots according to the TdT reaction time (0, 0.5, 1, 2, and 4 h) were 48.37 ± 28.16, 101.49 ± 26.33, 164.35 ± 34.42, 183.06 ± 26.88, and 177.3 ± 26.32, respectively. Two hours was determined to be the optimal TdT reaction time. The grey value of the microarray spot increased with time and showed a tendency to become saturated at 2 h (Fig. 3A). The mean grey values of spots according to b-dUTP concentration (0, 40, 80, 120, 160, and 200 µM) were 20.91 ± 1.17, 35.16 ± 9.28, 61.72 ± 17.82, 121.34 ± 8.46, 93.99 ± 25.53, and 110.19 ± 30.29, respectively. Therefore, a concentration of 120 µM led to the highest grey value and was determined to be optimal (Fig. 3B). In addition, the optimal concentration of the s-AuNP solution necessary to produce a colorimetric signal was also investigated, and 0.3 OD was determined as the optimal concentration, as shown in Fig. 3C. The mean grey values of spots according to s-AuNP concentration (0, 0.1, 0.2, 0.3, 0.4, and 0.5 OD) were 22.78 ± 0.57, 95.12 ± 10.8, 139.92 ± 10.12, 193.92 ± 22.74, 168.05 ± 28.67, and 137.55 ± 28.01, respectively. One reason the grey value decreased as the concentration of s-AuNP increased in this result could be due to electrical repulsion between s-AuNPs. In our previous study [28], the surface charge of s-AuNP was confirmed to be – 24.8 mV, and the biotin-labeled poly uridine tail generated from the array spot will also be negatively charged by the sugar-phosphate backbone. Therefore, excessive s-AuNP concentration conditions can inhibit the s-AuNP binding efficiency to the array spot due to the increased electrical repulsion between particles. To form the highest colorimetric signal at the array spot, the optimal condition was briefly set to a TdT reaction time of 2 h, b-dUTP concentration of 120 µM, and s-AuNP concentration of 0.3 OD. Captured images of the array spots formed on the COC chip was used for the characterization in Fig. 3 (Additional file 1: Fig. S2).

Fig. 3
figure 3

Optimization results for TdT and s-AuNP hybridization reactions

Usability of the proposed method

The applicability of the proposed method for actual pathogenic gene analysis was evaluated. First, to produce target dsDNAs from four kinds of microbial culture mediums, a PCR was performed using the four types of target-specific forward primers and reverse primers modified with a 5ʹ phosphate group. Subsequently, PCR products were treated with lambda exonuclease such that only ssDNAs capable of hybridizing with each capture probe DNA remained (Additional file 1: Fig. S3). These test samples were individually prepared in the presence or absence of each pathogen and analyzed on a COC chip to which each target-specific capture probe DNA was immobilized. As shown in Fig. 4A, it was visually apparent that the colorimetric signal of the microarray spots enhanced remarkably in the presence of each target ssDNA for all pathogens. In addition, the average grey values of spots obtained by analyzing Fig. 4A through Image J software are 42.74 ± 6.98 for E. coli (0 cell), 180.31 ± 20.38 for E. coli (105 cell), 60.97 ± 7.66 for Listeria (0 cell), 182.04 ± 14.5 for Listeria (105 cell), 66.28 ± 14.16 for Bacillus (0 cell), 202.8 ± 19.26 for Bacillus (105 cell), 57.42 ± 6.31 for Salmonella (0 cell), and 187.44 ± 26.29 for Salmonella (105 cell) (Fig. 4B). These results suggest that the present detection method could easily help identify the presence or absence of each pathogenic gene from several pathogen samples with the naked eye.

Fig. 4
figure 4

A Grey capture images of array spots formed on the COC chip to detect four pathogenic genes. Scale bar is 4 mm. B Bar graph of the grey values of (A) images analyzed using Image J software. The symbol (− or +) indicates the amount of each pathogen (0 or 105 cells)

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