Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores
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This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 × 105 Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use.
KeywordsBiochips Biosensors immunoassays ELISA Spectroscopy Bacillus globigii Chemiluminescence Spores Intensifier Bioluminescence
Over the last decade, there has been a growing interest in the rapid detection of pathogens for homeland defense [1, 2, 3]. Of particular concern among the potential biological warfare (BW) agent candidates for use as a weapon is Bacillus anthracis for a number of reasons. For example, B. anthracis can be produced and released in large numbers in the spore form, which is highly resistant to inactivation. Moreover, it is a highly pathogenic organism, requiring medical attention within 24–48 h of initial exposure, and infective doses have been estimated to be very low (e.g., 8,000 to 10,000 spores inhaled) . Consequently, there is a definite need for practical detection devices capable of identification and quantitation of BW agents, such as B. anthracis spores.
One of the primary approaches for detection and species-specific identification of BW agents is based on immunological recognition [2, 4, 5, 6, 7, 8]. In addition to being both highly sensitive and selective, immunological techniques can also be easily adapted to field use [2, 9, 10, 11]. Unlike nucleic acid-based analyses, immunological methods do not require a cell/spore lysis step for the extraction of DNA or RNA, as surface antigens can be targeted. This is particularly advantageous for the detection of bacterial spores, such as B. anthracis, as it is very difficult to disrupt the strong, resistant shell of the spore form .
Development of a method incorporating a chemiluminescent product allows for a number of potential advantages over fluorescent methods including extreme sensitivity, as well as simpler instrumentation [5, 15, 16, 17, 18]. More specifically, with chemiluminescence, photons are generated only when the reactants are present, unlike fluorescence where an excitation source could induce nonspecific radiation from either scattering or background excitation of the sample matrix. Since problems of light scattering, nonselective excitation, and source instability are absent, chemiluminescence possesses an inherently low background, allowing for a very sensitive analysis. Moreover, since an excitation source and associated optics required for fluorescence analyses are eliminated, detection can be achieved with a simpler system that is readily adaptable to field use. Previously, we have developed an integrated circuit biochip that has demonstrated great potential for field use . This biochip device has a number of distinct advantages over alternate biosensing technologies including a fabrication process based on complementary metal oxide semiconductor (CMOS) technology and multianalyte detection [6, 13, 19, 20, 21]. For example, the CMOS fabrication process allows for application-specific circuitry (i.e., signal amplification and filtering) to be integrated into the chip, thereby significantly reducing the size and power requirements of the system. Another important consideration is that the CMOS process is very cost-effective, which is ideal for mass producing portable detection devices. Furthermore, the chip is composed of an array of individual detector elements, each of which could be devoted to the detection of a different biological agent for multiplexed detection. For example, in this work a 4 × 4 photosensor array was used, which could be capable of performing 16 simultaneous biowarfare agent analyses in a single, compact unit.
In the current work, the first biochip device with an integrated intensifier enabling chemiluminescence detection is reported and the feasibility of using this system for the detection of Bacillus globigii spores is demonstrated. This system has a number of potential advantages over previous designs, including elimination of a laser and associated optics used for sample excitation in fluorescence systems. Furthermore, this simpler design, along with the self-contained biochip integrated circuit, could allow for the development of a small yet sensitive system for field use.
Intensified biochip detection system
The integrated circuit biochip prototype was developed previously by the Vo-Dinh group at Oak Ridge National Laboratory . Each photosensor is 900-μm square in size and is arranged in a 1-mm grid array. Through a 1.2-μm n-well CMOS fabrication process, application-specific circuitry was integrated into the chip for digital control of signal filtering and amplification. The resulting output voltage from each of the individual photosensors was recorded using a laptop computer with in-house written Labview software. The chemiluminescence signal intensity, originating from the 16 locations, was then correlated to the concentration of target B. globigii spores.
ELISA procedure for detection of B. globigii spores
An enzyme-linked immunosorbent assay (ELISA) for antibody-based capture and identification of Bacillus globigii (B.g.) spores was used in conjunction with the biochip detection instrumentation. As illustrated in Fig. 1, antibodies specific to a surface antigen on the B.g. spore (goat anti-B. globigii diluted to 10 μg mL−1 in 0.1 M carbonate buffer, pH 9.6) were immobilized onto a Nunc maxisorp protein binding platform (Nunc Maxisorp 96-well plate surface) overnight at 4 °C. For all other antibody binding steps, incubation times were approximately 1 h in order to insure complete binding. However, these times could be reduced significantly for field use [6, 14].
After immobilization of the capture antibodies, the remaining binding sites were blocked for 1 h at room temperature using a bovine serum albumin (BSA) diluent/blocking solution concentrate, diluted 1:10 in distilled water (Kirkegard and Perry Laboratories (KPL), Gaithersburg, MD). Following blocking, the immobilized antibodies were then incubated with Bacillus globigii spores (Var. Niger, Baker Labs Dugway Proving Grounds, UT) diluted to various concentrations in phosphate-buffered saline (PBS) at 37 °C from a solution in the same buffer. Bacillus globigii stock solution concentration was determined by both serial dilutions and standard plate counts grown on a generic growth medium (trypticase soy agar) as well as through counting using a hemocytometer. Following incubation with the spores, the wells were washed thoroughly in PBS + 0.5% Tween 20 to remove any unbound target species. Subsequently, a detector antibody (rabbit anti-B. globigii), recognizing another epitope on the B.g. spore surface, was diluted in BSA diluent/blocking solution concentrate (1:15 dilution in distilled water) to a final concentration of 5 μg mL−1 and incubated at 37 °C with the captured spores before being washed several times (PBS + 0.5% Tween 20). The final antibody, goat anti-rabbit IgG (H+L) conjugated with alkaline phosphatase (Jackson Immunoresearch Laboratories, Avondale, PA), was diluted 1:3,000 in alkaline phosphatase (AP) stabilizer (KPL, Gaithersburg, MD), and was incubated at 37 °C with the sample complex. The unbound enzyme–antibody conjugate was then removed through several washes as described above. Finally, 100 μL of the chemiluminescent substrate based on a novel 1,2-dioxetane (Intergen Bold APS 540) was incubated with the immunocomplex for 1 h to yield a detectable chemiluminescent product emitting 540-nm light. For all experiments here, the reactions were carried out under “saturated substrate” conditions. The results from three separate bioassays were recorded ten times each per chamber and averaged for each of the data points described below. The error bars shown on the graphical results represent plus or minus one standard deviation calculated from these replicate assay experiments and measurements.
Results and discussion
One of the greatest challenges to biowarfare detection is the development of field-deployable instrumentation capable of performing sensitive analyses. Not surprisingly, current research efforts of many groups aim to improve detection capabilities by incorporating some form of amplification of either the target (e.g., polymerase chain reaction for DNA targets) or indirect amplification of the signal (e.g., enzyme-linked immunosorbent assay for protein targets). Another method of achieving signal amplification is by enhancing the capabilities of the detection device by using an intensified system. However, with a typical fluorescence-based analysis, background photons originating from nonspecific excitation and emission or light scattering of the light source will also be amplified by the intensifier.
Intensified biochip system for chemiluminescence detection
Detection of Bacillus globigii spores using the intensified biochip system
This method produced little background levels, as evidenced by the comparable signal intensity of the negative control relative to that observed for the substrate. One possible source of this low background includes nonspecific interactions of either the rabbit anti-B.g. or alkaline phosphatase-conjugated goat anti-rabbit detector antibodies used in the immunoassay as described in Fig. 1. Consequently, it is possible that the low background levels and error bars produced in these studies could be further reduced through systematic optimization of the blocking agent, reagent concentrations, and washing conditions, for minimal nonspecific interactions and maximum assay reproducibility.
These results show that enzyme-based amplification offers a potentially attractive alternative to nucleic acid-based amplification methods (e.g., polymerase chain reaction), since a sensitive analysis can be performed without requiring a cell-lysing step that is particularly difficult to achieve with the hard, resistant shells of spores. In addition, the ELISA method utilizing a chemiluminescent product is characterized by an extremely low background, which couples well to the compact intensified biochip.
This is the first report of an intensified biochip system for the detection of biological agents. Through the combination of the sensitive intensified biochip system ideal for low-background chemiluminescence measurements, and the enzyme-based signal amplification, detection of 5 × 105 spores was demonstrated. Advantages of this system include elimination of a laser and associated optics used for sample excitation in fluorescence systems, and a chemiluminescence-based method characterized by an inherently low background, which is ideally suited for an intensified device. Furthermore, the simple design, along with the self-contained biochip integrated circuit, could allow for the development of a small yet sensitive system for field use. Moreover, multiple biowarfare agents could be detected within a single, compact unit, as each biosensing element could be devoted to the detection of a different biological agent.
This research was sponsored by the U.S. Department of Energy, Chemical and Biological National Security Program, under contract DE-AC05-00OR22725 with UT-Battelle, LLC. The authors also thank Dr. Arpad Vaas (Oak Ridge National Laboratory) for his help in the preparation of the Bacillus samples used in this work.
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