- Nano Express
- Open Access
Capillary-driven surface-enhanced Raman scattering (SERS)-based microfluidic chip for abrin detection
- Hao Yang†1,
- Min Deng†2,
- Shan Ga†1,
- Shouhui Chen2,
- Lin Kang1,
- Junhong Wang1,
- Wenwen Xin1,
- Tao Zhang1,
- Zherong You1,
- Yuan An1,
- Jinglin Wang1Email author and
- Daxiang Cui2Email author
© Yang et al.; licensee Springer. 2014
- Received: 19 February 2014
- Accepted: 11 March 2014
- Published: 24 March 2014
Herein, we firstly demonstrate the design and the proof-of-concept use of a capillary-driven surface-enhanced Raman scattering (SERS)-based microfluidic chip for abrin detection. The micropillar array substrate was etched and coated with a gold film by microelectromechanical systems (MEMS) process to integrate into a lateral flow test strip. The detection of abrin solutions of various concentrations was performed by the as-prepared microfluidic chip. It was shown that the correlation between the abrin concentration and SERS signal was found to be linear within the range of 0.1 ng/mL to 1 μg/mL with a limit of detection of 0.1 ng/mL. Our microfluidic chip design enhanced the operability of SERS-based immunodiagnostic techniques, significantly reducing the complication and cost of preparation as compared to previous SERS-based works. Meanwhile, this design proved the superiority to conventional lateral flow test strips in respect of both sensitivity and quantitation and showed great potential in the diagnosis and treatment for abrin poisoning as well as on-site screening of abrin-spiked materials.
- Capillary force
- Microfluidic chip
Recently, ricin has caught the public's attention by the toxin-tainted letters sent to US President Barack Obama, Mississippi Senator Roger Wicker, and a Mississippi justice official, while abrin, its 70 times more toxic analogue, is less known to the general public. Abrin and ricin are toxic proteins with similar structure and properties, both of which are classified as category B select agents by the US Health and Human Services . Compared with ricin, abrin is much more poisonous with an estimated human fatal dose of 0.1~1.0 μg/kg . Although there are reported deaths on account of intentional poisoning, most cases occur in children by unintentional ingestion . After ingestion, the major symptoms of abrin poisoning may occur in less than 6 h, and the deaths in children dying of ingestion of one or more abrin seeds have been documented in literature . Therefore, a fast, readily available confirmatory testing will greatly facilitate the timely diagnosis and treatment for abrin poisoning.
Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that provides a highly enhanced Raman signal from Raman-active molecules that have been adsorbed onto rough metal surfaces. The reported surface enhancement factor ranges from 103 to 1015, which means that the technique may detect proper analytes at a single molecule level [5–8]. There are two effects, chemical and electromagnetic, attributing to the enhancement. The former involves the formation of a charge-transfer state between the metal surface and adsorbate, contributing 1 to 2 orders of magnitude to the overall enhancement, while the latter is the dominant effect, arising from the collective oscillation of conduction electrons due to the irradiation of a metal by light . Besides high sensitivity, the Raman scatter possesses 10~100 times narrower bands than those of fluorescence and excellent anti-photobleaching properties, which avail to reduce undesirable spectral overlap and provide long and stable signal readout . So far, there have been many different SERS-based analytical techniques that have been developed for cancer markers, infectious diseases, pH sensing, etc. [8–15]. These techniques unleash tremendous potential for ultrasensitive biomedical analysis. However, it still remains a great challenge to reduce the overall cost while maintaining the advantages of sensitivity, because most SERS-based detection systems are strongly dependent on the relatively expensive process of microelectromechanical systems (MEMS), especially sputtering of a noble metal layer.
All animal experiments (No. SYXK2007-0025) were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University.
Extraction of natural abrin
Natural abrin was extracted according to the previous method with slight modifications . Briefly, the decorticated seeds of Abrus precatorius (approximately 100 g) were soaked in 200 mL of 0.01 M phosphate buffer solution (PBS) at pH 7.4 and 4°C for 24 h. After thorough homogenization, the puree was centrifuged at 10,000g for 30 min. Then, the aqueous layer was saturated with ammonium sulfate (95% w/v) and centrifuged at 10,000g for 30 min. The precipitate was dissolved in 100 mL of 0.01 M PBS and applied to a 1.5 × 10 cm Gal-agarose column (EY Laboratories Inc., San Mateo, CA, USA). The bound abrin was eluted with 0.125 M d-galactose solution. The collected fractions were dialyzed and applied to a Sephacryl S-100 prepacked column (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA) equilibrated in PBS. The as-prepared abrin was analyzed by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Preparation of anti-abrin polyclonal antibodies
The purified abrin was inactivated by formalin and used to hyperimmunize a rabbit, and 0.5 mL of abrin toxoid (80 mg/mL) was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously to the rabbit. Seven days later, immunization was carried out four times including one booster immunization with the mixture of the abrin toxoid and Freund's incomplete adjuvant as well as three injections with the toxoid at weekly intervals. Ten days after the final injection, the immunized blood was collected by jugular puncture, and the serum was separated for subsequent purification of anti-abrin polyclonal antibodies with rProtein A Sepharose Fast Flow (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The antibody titers were evaluated by enzyme-linked immunosorbent assay (ELISA).
Preparation of external SERS probes
The external SERS probes were prepared according to a published method . DTNB (5,5′-dithiobis (2-nitrobenzoic acid), Sigma-Aldrich Co. LLC, St. Louis, MO, USA) was used as the Raman-active tag. One milliliter of purified anti-abrin polyclonal antibodies (approximately 75 mg/mL in 0.01 M PBS) was dropwise added to 1 mL of 20-nm colloidal gold solution (Sigma-Aldrich Co. LLC) under stirring. After 1 h of incubation at 4°C, the antibody-coated colloidal gold was separated by centrifugation at 12,000g for 1 h. Bovine serum albumin (BSA) was used to block the unmodified colloidal gold at a final concentration of 0.5% (w/v). The labeled colloidal gold was centrifuged at 12,000g for 1 h and resuspended in 1 mL 0.01 M PBS solution. Twenty microliters of DTNB solution (1 mM in 0.01 M PBS) was added to the gold solution and incubated at 4°C for 1 h. The resultant SERS probes were centrifuged again at 12,000g for 1 h and then resuspended in 0.01 M PBS for later use.
Fabrication and surface modification of gold-coated silicon wafer
Assembly of capillary-driven chip
Anti-abrin polyclonal antibodies and goat anti-rabbit secondary antibodies (1 mg/mL) were dispensed on the gold-coated wafer with a Biodot XYZ3000 dispenser (Biodot Inc., Irvine, CA, USA) as test zone and control zone, respectively. After drying for 30 min, the wafer was blocked with PBS containing 1% BSA (w/v). The SERS probes were printed on a glass fiber filter as conjugate pad and dried at room temperature. The absorbent pad, conjugate pad, and sample pad were cut into strips of 1 mm in width with a guillotine cutter and overlapped on the laminating card with the dried wafer as shown in Figure 1.
SERS signal measurement
The purified abrin was diluted with a series of concentrations from 0.1 ng/mL to 10 mg/mL in 0.01 M PBS solution. Fifty microliters of the diluted toxin solution was added to the sample pad, and the SERS signal was read out with i-Raman-785S (B&W TEK Inc., Newark, DE, USA) after 5 min. The intensity of the peak at approximately 1,330 cm-1 was used to quantify the abrin in PBS solution.
Characterization of natural abrin and anti-abrin antibody
Characterization of microfluidic chip
Analysis of abrin-spiked sample
As previously mentioned, SERS-based techniques showed many potential advantages including high sensitivity, narrow bandwidths, and photobleaching resistance. It still remains a challenge to develop a SERS-based immunodiagnostic technique of both low cost and good operability. Some pioneering researchers have published their works focusing on the ultrasensitivity from the level of picograms per milliliter to femtograms per milliliter [6, 8, 9, 11, 14, 23–28]. Compared with their work, our design strategy emphasized the operability of SERS-based technique. In other words, this strategy is aimed at not just a comparative LOD, but a balanced solution between the complication of new techniques and the universality of traditional ones.
We have designed and demonstrated the proof-of-concept use of our capillary-driven SERS-based microfluidic chip for abrin detection. The microfluidic chip was fabricated by MEMS process. The combination of the traditional LF test strip with capillary-driven gold-coated substrate results in the enhancement of sensitivity as well as the reduction of cost for SERS-based immunodiagnostic techniques. In this work, a calibration curve was obtained to detect the concentration of abrin in the range from 0.1 ng/mL to 1 μg/mL, which is superior to the traditional LF test strip for the same purpose in respect of both sensitivity and quantitation . What is critically important is the operability of our design strategy, that is, the performance of traditional LF test strips is improved without excessive increase in complication and cost of fabrication. In addition, this SERS-based microfluidic chip can be further developed and applied to other on-demand and point-of-care detection for a substance of interest.
This work is supported by the National Key Basic Research Program (973 Project) (No. 2011CB933101), National Natural Scientific Fund (Nos. 81225010, 81327002, and 31100717), 863 project of China (2012AA022703), Shanghai Science and Technology Fund (Nos. 13NM1401500 and 11nm0504200), and Shanghai Jiao Tong University Innovation Fund for Postgraduates (No. AE340011).
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