Nucleic acid is the basic genetic material of creatures and plays an extremely significant role in the growth, reproduction and inheritance of creatures [1
]. Nucleic acid detection is of great significance and demand in clinical diagnosis, environmental monitoring and food safety [3
]. Currently, including polymerase chain reaction (PCR) and other isothermal amplification technologies, DNA microarray technologies are conventional methods for nucleic acid detection [6
]. The detection method based on PCR is time-consuming and complex to operate. In addition, this method cannot be used for real-time detection [9
]. DNA microarray technology is stringent for technical personnel. It requires expensive instruments and complex preparation of samples. Droplet digital PCR is a new method developed on the basis of conventional PCR. It has the advantages of absolute quantification and high sensitivity. Its lower detection limit is lower than that of conventional PCR technology. However, droplet digital PCR relies on expensive instruments and costs a lot, and so its popularity is not high at present [10
]. Compared with the mentioned techniques, nucleic acid detection based on surface plasmon resonance (SPR) sensing technology is a label-free, real-time, simple to operate, and highly sensitive detection method [11
]. SPR sensing technology has been already used to study nucleic acid interactions.
SPR, as a physical optical phenomenon occurring between the metal film and thinner medium on its surface, is very sensitive to the change of refractive index of the media close to the surface of the metal film [16
]. The position of the resonance absorption peak changes with the refractive index of the medium, which is an inherent feature of all materials. By using this principle, the corresponding material concentration and dynamic characteristics can be detected [18
]. Compared with traditional biochemical analysis technology, SPR sensors have been rapidly developed due to their excellent characteristics of real-time monitoring, label-free, less sample consumption and highly sensitive quantitative detection [19
]. At present, SPR sensing technology has become a mature method for detection of biomolecular interactions. It has been applied in many fields, such as clinical disease diagnosis, environmental monitoring and quantitative detection of transgenic ingredients, and so on [20
]. SPR sensing technology can detect small changes in the refractive index effectively with high sensitivity. However, in order to detect even smaller changes in the refractive index, amplification markers such as liposomes, latex particles and proteins need to be used for further sensitivity improvement [21
In addition to biological methods to improve the detection sensitivity, different optical detection modes of SPR will also affect the sensitivity. The methods of SPR sensing technology mainly include angle modulation, wavelength modulation, phase modulation and intensity modulation [23
]. Among them, SPR angle modulation method can obtain a large refractive index measurement range, and its measurement sensitivity is very high, up to 1 × 10−7
]. However, angle modulation usually requires a high-resolution stepper motor and mechanical to adjust the angle. The complex structure and large volume of this instrument limited the development of point-of-care testing (POCT).
Currently, most commercial SPR instruments which adopt Kretchman prism-coupled optical structure are expensive to purchase and maintain, large in size, complex in structure, and professional services in need [25
]. It brings great challenge to the popularization and practical application of SPR instrument. Therefore, miniaturized and portable SPR instruments is the development tendency. Pan et al. developed a portable SPR instrument which has a size of 550 mm × 200 mm × 330 mm, an angle scanning accuracy of 0.002°, a refractive index resolution of 10−6
RIU, and an angle scanning range of 64°–84°. It has been successfully applied to the detection of hemocyanin and microcystin in shrimp [26
]. Laksono et al. developed a low-cost, high-precision SPR sensing system that applied anti-backlash gear to angle modulation. This kind of anti-backlash gear structure improves the system’s angle scanning range to 30°–70°, and the accuracy is 0.01° [27
]. There are also studies using 3D printer technology to manufacture SPR parts to reduce costs. For example, the mechanical alignment components in the SPR instrument developed by Manjunath were made using 3D printing technology. Manjunath developed low-cost spectrometers using diffraction gratings and webcams. He built a dual-channel SPR system with a sensitivity of 10−4
RIU using two plastic fibers [28
]. Zhang et al. used 3D printing technology of molten bulk molding to make some mechanical parts required by the system to reduce the cost and shorten the development cycle. The angle scanning range of the SPR system can reach 40°–72°, and the resolution measured by the system is 8.34 × 10−6
]. There are also studies that have combined the versatility of smart electronics with the sensing capabilities of SPR technology. For example, Vestri used the Raspberry Pi single board computer equipped with touch screen to realize the management of the entire measurement process of SPR. Vestri used the CCD image sensor as the signal acquisition, so that the refractive index resolution could reach 4.9 × 10−6
]. In the above studies, some angle scanning structures were complex, requiring the use of two stepper motors to complete a wide range of angle scanning. Some could not achieve angle scanning. None of these studies achieved an effective combination of high precision, wide range of angle scanning and high detection sensitivity. Therefore, it is necessary to develop an SPR sensor with high precision, wide angle scanning range and high sensitivity.
There have been many studies on SPR sensors for nucleic acid detection. Kim et al. employed a SPR method for the quantitative detection of avian influenza DNA hybridization. The sensor has a refractive index resolution of 1.6 × 10−4
RIU. The incident angle of the sensor is fixed at 60°. They used wavelength modulation of the sensor to detect target DNA of 1 μM. The incident angle of this sensor is fixed so that it cannot achieve angle modulation [31
]. Ding et al. completed highly sensitive detection of microRNA using SPR biosensors. The target miRNA was first captured by the DNA probes on the surface of the chip, and then streptavidin was employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies. This signal amplification method made the sensitivity of the SPR biosensor very high. The biosensors could detect target miRNA as low as 9 pM in 30 min using the method [32
]. Bagi et al. implemented morpholino-based nucleic acid sensing on a portable SPR instrument. They tested and evaluated two signal amplification methods. A significantly higher response was observed with the sequential approach (up to 16-fold). The binding of the target oligonucleotide labeled with the biotin probe occurred on the sensor surface, and then the streptavidin magnetic beads were bound to the immobilized target. This method could detect a 45-nucleotide long oligo target as low as 0.5 nM (7.2 ng/mL) [33
]. Therefore, the methods with high sensitivity for nucleic acid detection adopt the method of biological signal amplification, while for the direct method, the limits of detections are in the nanomolar range, which are weaker than sensitivity-enhancing methods [34
]. However, the process of direct detection is simple and convenient for real-time and fast detection in field use.
In this paper, a portable multi-angle scanning SPR sensor was designed. The angle modulation of the sensor uses the mechanical scanning method. It only uses one stepping motor to rotate and drive a belt. The belt pulls the mechanical linkages of incident light and reflected light to adjust the length and angle of the linkages at the same time. The intermediate prism is fixed so that the incident angle is always equal to the reflected angle. The angle scanning accuracy of the sensor is measured to be up to 0.002° and the resolution of the refractive index to be up to 3.72 × 10−6 RIU. The sensor can control the angle of incident light and reflected light by the motor to achieve real-time monitoring. Finally, the nucleic acid hybridization experiment was performed on a gold film chip modified with BSA. The experimental result showed that the instrument had good performance and could achieve nucleic acid hybridization detection effectively.
2. Materials and Methods
2.1. Surface Plasmon Resonance (SPR) Sensor Setup
The portable multi-angle scanning SPR sensor includes: a horizontal drive module, symmetrical optics module, and prism and flow cell module. As the motion system of SPR sensor, the horizontal drive module includes a motor, belt wheel, belt, sliding block and guide rail. The two sliding blocks are respectively fixed on the upper surface and the lower surface of the belt. The sliding blocks can generate symmetrical motion along the guide rail under the belt drive. The two sliding blocks drive two linkages on each side of the incident and reflected light. One end of the linkages on each side is respectively fixed on the left or right sliding blocks and the other end is fixed on an axis of rotation as the center of rotation. The two linkages on each side are connected in parallel. The total length of the two linkages can be adjusted automatically so that the linkages of the left and right side produce symmetrical motion. The principle of angle modulation is presented in Figure 1
The portable multi-angle scanning SPR sensor only requires a stepper motor to drive the belt. The sliding block on the belt is used to pull the incident light linkages and reflected light linkages on the symmetrical optical module to move symmetrically. The prism position is fixed. This structure makes the incident angle equal to the reflected angle at all times. If the prism position is adjusted up and down, the angle detection range can be expanded. The incident angle of the sensor can change from 30° to 74°. The detection from the gas phase to liquid phase can be performed. The red laser light source (wavelength is 628 nm) is fixed on the incident light linkages. A standard photodiode power detector is installed on reflected light linkages (Note: fix the detector in a position where the light can be concentrated in the center of the photosensitive area). The photoelectric signal collected by the photodiode power detector (Thorlabs) is processed by the digital signal processing chip and then the signal is transmitted to the computer. The computer can display the relation graph of light intensity and time in real time. The SPR system can be operated by a laptop for field applications in this paper. The prism and flow cell module uses the classic Kretschmann prism structure. This module uses a triple prism (n = 1.72; Nanjing, China).
In practice, a glass slide sputtered with metal (usually gold) was attached to the top surface of a prism with matching oil. Then, the flow cell was balanced on the glass slide through the pressure structure. In order to prevent the interference of natural light, the sensor was designed with a light-proof structure. The overall size of the SPR sensor is 480 mm × 150 mm × 180 mm, and the total weight is 3.09 kg. The prototype SPR instrument is presented in Figure 2
2.2. SPR Chip and the Flow Cell
Two kinds of SPR sensor chip were used in this paper. The first chip was fabricated by magnetron sputtering technology on 20 mm × 20 mm × 0.17 mm glass slide. In this process, 2 nm of chromium and 50 nm of gold was sputtered in an area of 5 mm× 5 mm successively. Before the experiment, the metal surface was cleaned with a sterile cotton soaked in alcohol and then was dried. The second chip was made by modifying BSA on the first chip. The concentration of BSA solution used in the modification process was 2 mg/mL. It took 30 min to modify BSA on the metal surface. The second chip was used in the nucleic acid detection. The flow cell was made of PDMS oligomer and a crosslinking agent (Sylgard 184). The mixture of PDMS and Sylgard 184 with a 10:1 ratio was degassed under vacuum, poured into a silanized glass mold, and then cured in an oven at 80 °C for 8 h.
2.3. Materials and Samples
The reagents used in the sensor performance test were deionized water and glycerol solution with a 20% volume fraction. Different concentrations of glycerol were obtained by diluting a glycerol solution of 20% volume fraction with deionized water.
In the nucleic acid hybridization experiment, phosphate buffer salt (pH 7.4 PBS 10×, diluted by deionized water 10 times) was the buffer solution. N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were the activation solution. Ethanolamine was the closed solution, and 10 mM glycine-HCL was the regeneration solution. All the above solutions were purchased from GE Healthcare. Streptavidin (SA), DNA probes molecule (5′ - TTTCTACCCTTTGGTGCTAATGCCCATACT - 3′, 5′end with biotin), DNA (5′ - AGTATGGGCATTAGCACCAAAGGGTAGAAA - 3′) and non-complementary DNA sequence (5′ - TTTCTACCCTTTGGTGCTAATGCCCATACT - 3′) were kind gifts from the Institute of Biophysics, the Chinese Academy of Sciences.
2.4. Sensor Performance Test
After the SPR sensor was built, the angle scanning test was implemented with deionized water to test the practicability of angle scanning. During the test, a limit switch was used to return the angle of incident light to the origin that was the position of small angle. Then the angle was scanned from 44.438° to 59.849°. The scanning speed was 1.1607 × 10−2 m/s. The intensity signal of the reflected light was collected during the angle scanning. The signal acquisition frequency was 4.61 Hz.
The refractive index resolution of the instrument was measured by means of intensity modulation. No.0–No.10 glycerol was prepared and the volume fraction interval was 1%. No.0 was deionized water, No.1 was 1% glycerol, No.2 was 2% glycerol, and so on.
2.5. Experimental Protocol
There were four steps in the DNA detection experiment shown in Figure 3
In step 1, a gold film chip was modified with BSA. Then, the modified chip and the flow cell were installed on the triple prism.
In step 2, PBS buffer solution was injected into the flow cell for 10 min to clean the surface of the chip. Then, the BSA molecules on the chip surface were activated by injecting the activation solution for 8 min. PBS buffer solution was then injected for another 6 min. Next, SA diluted 25 times with PH4.5 sodium acetate was injected into the flow cell for 10 min. SA was bound to the activated BSA. After that, PBS buffer solution was injected for 6 min to remove the free SA molecules.
To optimize the fixed quantity of SA molecule, SA diluted 10 times with PH4.5 sodium acetate was injected into the flow cell for 5 min. PBS buffer solution was then injected for 6 min to wash the surface, and ethanolamine was injected for 8 min to block the unbounded activation sites. This could prevent nonspecific binding in subsequent experiment.
In step 3, PBS buffer solution was injected for 5 min, followed by a solution of DNA probes (50 nmol/mL) for 5 min in order to bind the DNA probes to SA. PBS buffer solution was then injected for 6 min to wash the free DNA probes molecules.
To optimize the fixed number of DNA probes, a solution of DNA probes (1 μmol/mL) was injected for 7 min.
Finally, in step 4, DNA detection hybridization experiment was implemented by injecting 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 μmol/mL of DNA solution successively. After each test of a concentration of DNA solution, 10 mM glycine-HCL was injected for regeneration.
In this paper, by combining it with the classical Kretschmann prism structure, a portable multi-angle scanning SPR sensor with a simple angle scanning method and symmetrical optical path is developed for nucleic acid hybridization detection. The sensor is of high precision, simple control, and wide range of angle modulation. In addition, the resolution of angle scanning can reach 0.002°. In addition, the mechanical structure of the sensor is simple, stable and miniaturized, which is easy to carry and install. More importantly, it can greatly reduce the cost and facilitate mass production, thus accelerating the popularization and application of SPR sensor devices.
The sensor was used to implement sensitivity tests and hybridization experiments between DNA solutions in different concentrations and DNA probes. The results show that the SPR sensor has a high sensitivity of 3.72 × 10−6 RIU. The sensor has a fast response speed, and can successfully accomplish the hybridization detection of DNA with a solution of 0.01 μmol/mL.