Abrin from Abrus precatorius
is a highly toxic ribosome inactivating protein. It has similar characteristics to ricin, which has been used as a terrorist threat, but more toxic [1
]. As a category B agent of potential bioterrorism risk by the “Centers for Disease Control and Prevention Moran” [3
], abrin’s detection and prevention has become a focus of the public security field. Currently, the methods to detect abrin mainly include enzyme-linked immunosorbent assay (ELISA) [4
], radioimmunoassay [6
], immunochromatography [7
], piezoelectric methods [8
], some molecular biological methods [9
] and spectroscopic methods [10
]. These methods have enriched the detection systems of abrin, but more or less possess several weak points such as complicated operation, long detection time or low sensitivity. As a rising technique in recent years, electrochemiluminescence (electrogenerated chemiluminescence (ECL)) sensing method has also been used for testing abrin, and obtained a good result with the LOD of 0.1–0.5 ng/mL [5
The ECL sensor has extensive advantages, such as high sensitivity, wide detection range, good controllability, fast response and strong anti-interference ability and so on, which make it especially suitable for the detection of trace and ultra-trace amount of complex substances [11
]. However, traditional ECL detection equipment based on magnetic separation technology needs to integrate some necessary components including discrete three-electrode system (working electrode, counter electrode and reference electrode), ultra weak photoelectric detection system, focusing lens system, magnetic separation and enrichment system and micro channel sampling and cleaning system into a unified ECL detection pool, which brings it some defects of high price, bulk and weight, inconvenient cleaning and so on. All of these have hindered the popularization and application of ECL sensor to a certain extent. To solve these problems and achieve in-situ, point-of-care testing, many novel designs have been proposed such as Lab-on-a-paper immunodevices [20
] and microfluidic platforms [19
]. In these simplified and efficient devices, a screen-printed electrode (SPE) often serves as an important, or indispensable, component.
SPE is prepared by screen printed technology. It can be produced in batches with a low price and it is miniature and portable with a good electrochemical performance [21
]. Meanwhile, the SPE can be freely loaded and disassembled, bringing great convenience to the surface cleaning and modification of electrode, and the background interference caused by the inadequate cleaning of the traditional ECL reaction pool can be avoided. SPE highly integrates the three discrete electrodes. Used as a micro reaction pool, it can promote the miniaturization of ECL sensor and then enable environmental pollutants, biological warfare agents to be measured in situ. The portable ECL sensor based on SPE has broad application prospects in developing miniaturized, integrated and intelligent field inspection equipment.
In this study, the SPE is introduced into biotoxin detection by electrochemiluminescence immunoassay. Taking the virulent abrin as the target, and fully combining the advantages of SPE and magnetic separation immunoassay, we developed a portable ECL sensing system and establish a new method of ECL detection of biotoxin with high sensitivity and simplified operation, which can provide technical basis and reference for the outdoor environmental monitoring, food hygiene inspection, anti-bioterrorism and so on.
2. Materials and Methods
2.1. Reagents and Instruments
Ru(bpy)32+-NHS ester, Biotin-NHS ester and DMF (N,N-Dimethylformamide) were purchased from Sigma-Aldrich (Munich, Germany). M-280 Streptavidin coated magnetic microspheres was purchased from Invitrogen Life Technologies (Oslo, Norway). Bovine serum albumin (BSA) was purchased from Shanghai Sinopharm Group Co., Ltd. (Shanghai, China) and Human IgG was purchased from Beijing Biosynthrsis Biotechnology Co., Ltd. (Beijing, China). Ricin, abrin, pcAb and mcAb of abrin were all prepared in our lab. DMSO (Dimethyl Sulphoxide) was purchased from Beijing Xingjin chemical plant (Beijing, China). The Procell solution mainly containing TPA (Tripropylamine) was purchased from Beijing Biolot Diagnostics Co., Ltd. (Beijing, China) and the deionized water was used as the experimental water.
Screen-printed gold electrode (BVT Technologies, a.s., Czech; Figure 1
) was the ECL reaction center. Portable ECL immunoassay sensor was jointly developed by our laboratory and Xi’an Remex Analysis Instrument Co. Ltd. (Xi’an, China). Samples were incubated by HS-3 vertical mixer (Scientz Biotechnology Co., Ltd., Ningbo, China). Preparation of biotinylated antibodies needed dialysis bag and RCT heating magnetic stirrer (IKA Company, Staufen, Germany). Magnetic separation operation was carried by Magnetic Separation Rack (Promega Company, Madison, WI, USA). A280nm
values and absorbance spectrum were determined on BioMATE 3S UV-Vis spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Ru(bpy)32+
-labeled antibody was prepared by 1-15P centrifuge and VIVASPIN 500 ultrafiltration centrifuge tube (Thermo Fisher Scientific Inc., Waltham, MA, USA).
2.2. Design and Development of Portable ECL Sensing Platform
The portable ECL sensing platform was jointly developed by our laboratory and Xi’an Remex Analysis Instrument Co. Ltd. (size: 18.2 cm × 14.0 cm × 5.2 cm, volume: 1.325 dm3
, mass: 1.45 kg). In the design of this platform, the key point is to combine the photomultiplier tube (PMT) with SPE together organically, to solve the conundrum of integrated design of mechanical, circuit control, reaction driving, data acquisition problems and so on. Figure 2
indicates the general structure of portable ECL sensor (Figure 2
A,B) and SPE clamping mechanism (Figure 2
C). SPE and the clamping mechanism are supported by lead rail, they can move along the rail to the outside of the container with the assistance of the spring; The clamping mechanism is located at the hatch where are looped grooves which can attenuate natural light; PMT is fixed above the electrode surface. When the electrode and the clamping mechanism are completely moved into the container, the PMT’s light window is perfectly aligned with the luminous region on the electrode, enabling the sensor to collect signals with high efficiency.
There are two work modes for the portable ECL sensor: “independent work mode” and “online working mode”. The “independent work mode” means the sensor could perform a site survey in the absence of vehicle power (or electric supply) in the wild environment. It can detect, store information, display and print the result relying on the built-in rechargeable battery, SPE, internal storage, monitor and portable printer. When the sensor is in the “online work mode”, there is enough power supply (vehicle power or electric supply) and the sensor is connected to the computer. Utilizing the full-featured management software for detection and data exchange, more detailed test results could be output.
2.3. Experimental Method
Referring to methods from Liu [26
] and Mu [27
], this experiment mainly includes the following operations.
2.3.1. Preparation of Capture Probe
pcAb was used to prepare magnetic microsphere capture probe. First, the pcAb needed to be biotinylated: 3.5 mL pcAb solution (1 mg/mL) was mixed with 0.5 mL DMF solution of activated biotin (biotin-NHS ester, 1 mg/mL), and stirred by magnetic force at room temperature for 3 h. Then, the reaction mixture was dialyzed overnight at 4 °C with 0.05 M PBS as the dialysis solution, and the purified antibody was stored at −20 °C for use.
Then, to immobilize the biotinylated antibody on the magnetic microspheres: 400 μL streptavidin coated magnetic microspheres was washed by 0.01 M PBS (PH = 7.4) adequately, and then 1 mL PBS and 200 μL biotinylated pcAb were mixed after magnetic separation, and incubated with moderate rotation at room temperature for 1 h. After the reaction was completed, the supernatant was obtained by magnetic force for absorbance detection to determine the binding amount of the capture antibody. Subsequently, the precipitate was suspended with 0.01 M PBS buffer and discard the supernatant by magnetic separation. This process was repeated five times to remove residual resistance and other impurities thoroughly. Finally, the precipitate was resuspended in 0.01 M PBS buffer and stored at 4 °C for use.
2.3.2. Preparation of ECL Labeled Probe
Equal ratio DMSO and deionized water were used to prepare Ru(bpy)32+-NHS ester solution (10−3 mol/L). Then, 200 μL was drawn to mix with 200 μL mcAb (2 mg/mL) and 600 μL carbonate buffer (0.05 M, PH = 9.6). The mixture was incubated for 12 h in the darkness, and then centrifuged at 8000 g for 10 min followed by cleaned with PBS for three times. Finally, the labeled antibody was resuspended in 0.01 M PBS buffer and stored at −20 °C for use.
2.3.3. Determination of Abrin
Fifty microliters of capture probe and 50 μL abrin solution were mixed and incubated for 20 min at room temperature. After magnetic separation cleaning twice, 20 μL ECL labeled probe (100 μg/mL) and 80 μL 0.01 M PBS were added and incubated for 20 min [22
]. Then, the mixture was cleaned five times, and 50 μL Procell solution was added into the precipitate for ECL detection. Five microliters of magnetic microspheres were absorbed and applied evenly to the surface of screen-printed gold electrode each time, and the ECL intensity was observed by cyclic voltammetry. The test process is illustrated in Figure 3
The SPE has several typical characteristics of simple preparation, low cost, miniaturized and good electrochemical performance. Taking SPE as the reaction center, the authors built a portable ECL sensing platform, possessing some outstanding features such as simplified operation, miniaturization and portability. In this experiment, based on the portable sensor and magnetic separation immunoassay technique, a specific sandwich model was built and a successful detection of abrin was finally achieved. The LOD was 0.1 ng/mL and the quantitative range was 0.5–1000 ng/mL. The portable sensor exhibited high sensitivity, good reproducibility and excellent specificity, achieving a fine result better than the conventional ECL sensor. Subsequently, based on the feature that SPE is easy for surface treatment, nanophase materials can be used to modify SPE to improve the electrochemical performance and ECL reaction efficiency, thus greatly improving detection sensitivity. Then, the portable ECL sensor would have greater potential for application in the fields of physiological and pathological examination, environmental pollution monitoring and biological terrorism prevention.