The portable BioDetector integrated (pBDi) allows the rapid, specific and sensitive identification of bioterrorism relevant toxins. Application of the ready-to-use Toxin Test Kits 1 and 2 allows the parallel detection of five different toxins among the group of botulinum neurotoxins, staphylococcal enterotoxins as well as the plant toxins ricin and abrin within 20 min in an automated process.

Introduction

Due to the potential risk of terrorist attacks using highly toxic or pathogenic biothreat agents (bacteria, viruses and toxins) there is a need for rapid, sensitive and specific detection of all kinds of biothreat agents in the field. In general, nucleic acid based detection platforms are applied for pathogen detection. However, PCR is unable to detect proteinaceous toxins and requires a clean sample demanding elaborate sample preparation. In contrast, immunoassay based techno-logies offer the possibility for universal detection of bacteria, viruses and toxins. Several techniques, including in vivo assays, mass spectro-metry and immunological methods, have been developed for detection of toxins. Most immunological methods are performed in a single test format based on sandwich enzyme linked immunosorbent assay (ELISA) principle.

Application Note #CBRNE - 1849597Simultaneous detection of bioterrorism relevant toxins using pBDi

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Authors

Christopher Pöhlmann, Thomas ElßnerBruker Daltonik GmbH, Leipzig, Germany

Keywords Instrumentation and Software

Toxin detection pBDi

Biochip pBDi Control

Immunoassay

However, detection of potential biological agents in environmental samples requires assays that can recognize multiple analytes simultaneously to reduce response time. Fluorescence based high density microarrays are commonly applied for toxin detection [1]. However, the instrumentation for optical signal read out is rather complex. In contrast, electrochemical biochips offer the possibility to construct portable instruments for clinical or environmental applications. The high sensitivity along with cost- and time effectiveness is another advantage of electrochemical biochips [2].

The pBDi employing sensitive electrochemical biochip technology for performing multiplex ELISA can be used for the detection of up to six biothreat agents in the field. The pBDi is based on the technology of the Bruker bench top device pTD (portable Toxin Detector), however, exhibiting new features such as integration in suitcase, battery mode, bluetooth connection to a ruggedized tablet PC as well as assays for bacteria, viruses and toxins identification.

In this application note, we introduce the pBDi for the simultaneous detection of toxins associated with biological warfare agents (BWA).

Experimental

Botulinum neurotoxins (BoNTs) were purchased from Metabiologics Inc. (Madison, USA), whereas staphylococcal enterotoxins A and B (SEA and SEB) were purchased from Sigma-Aldrich (Steinheim, Germany). Ricin and abrin were purchased from Toxin Technologies (Sarasota, USA). Protein concentrations of BoNTs, SEs, ricin and abrin were determined fluorometrically using the QubitTM quantitation platform and the QubitTM Protein Assay Kit (Invitrogen, Karlsruhe). Diluted toxin samples were prepared immediately before use.

Staphylococcus aureus DSM 19044 (SEB, SEG and SEI producer) was received from the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ, Braunschweig, Germany). Staphylococcus spp. were grown in 5 mL Tryptic Soy Broth (Carl Roth GmbH, Karlsruhe, Germany) at 37°C, 170 rpm for 18 hours. 1 mL samples were taken from cells in the stationary growth phase. The samples were centrifuged at 13,000 rpm for 10 min and 4°C. The supernatant was stored at -20°C until further processing. For biochip analysis of culture supernatants 5% (v/v) normal rabbit serum (Sigma-Aldrich, Steinheim, Germany) was added before measurement.

For biothreat agents detection the pBDi with the pBDi Control software and the pBDi Toxin Test Kits (Bruker Daltonics, Germany) were used. pBDi Toxin Test Kits contain all reagents (enzyme, substrate, neutralizer, detection antibody mix and decontamination solution), biochip cartridges and buffers necessary for toxin detection and subsequent decontamination. All buffers and consumables necessary for sample preparation are included in the pBDi Sample Preparation Kit.

Results

Workflow for sample preparation and toxin detection

The straightforward analysis starts with resuspension of a liquid or solid sample in sample buffer (Figure 1). Particles of sample matrix can block fluidic pathways or valves and result in decreased performance or malfunction of pBDi device. Several sample matrices exhibit acidic or basic pH decreasing toxin recognition by antibodies. Hence, samples were adjusted to neutral pH and filtered through a special syringe filter before application for toxin detection. The pBDi Toxin Test Kits 1 and 2 contain optimized reagents

Figure 1: General workflow for toxin detection.

Data analysis

Data acquisition

Samplepreparation

Resuspension of kit reagents

Principle of electrochemical sandwich ELISA

Figure 2: Principle of toxin detection using electrochemical ELISA technology (pAPG =p-aminophenyl-ß-D-galactopyranoside; pAP = p-aminophenol).

to perform multiplex electrochemical ELISA in a stable, easy to use lyophilized format. After measurement pBDi runs a self decontamination procedure for detoxification of sample port, waste and fluidics. The software pBDi Control performs automatic analysis of results obtained after toxin measurement and indicates toxin detection. Total sample-to-answer time including a minimal sample preparation step which can be performed by first responders in the field is less than 25 minutes (20 minutes analysis time plus 2-5 minutes sample preparation time).

Toxin detection with the pBDi

Rapid, sensitive and selective identification of several toxins in parallel is of utmost importance for health care and safety reasons. The pBDi is a self-contained identification platform for automated and specific detection of biothreat agents. The detection principle is based on an ELISA procedure. Capture antibodies immobilized on gold electrodes facilitate the specific binding of corresponding biothreat agents (Figure 2). Detection of bound biothreat agents is realized by application of a detector-antibody-enzyme conjugate and measurement of the electrical current of an enzymatic redox reaction. This sandwich architecture results in the immobilization of the reporter enzyme ß-galactosidase in the vicinity of the particular electrode. Then, enzymatic hydrolysis of p-aminophenyl-ß-D-galactopyranoside occurs and leads to production of electroactive substrate p-aminophenol leading to a detectable electrical signal. The detection event is strongly amplified in this system and allows sensitive biothreat agent identification in a short time period. First, the high turnover of

enzymatic reaction contributes to the signal amplification and second, a redox recycling of quinonimine to p-aminophenol, built into the experimental procedure, provides a second signal amplification [2]. The current correlates to the amount of target molecules captured by the antibodies. The whole identification procedure is realized in approx. 20 minutes. The Toxin Chips 1 and 2 allow the identification of five BWA associated toxins each.

Toxin Chip 1 enables detection of BoNT/A, BoNT/B, BoNT/F, SEB and ricin, whereas Toxin Chip 2 allows detection of BoNT/C, BoNT/D, BoNT/E, SEA and abrin (Figure 3). Two independent electrode positions are used for detection of each target substance. In addition, three unspecifically coated negative control positions for deriving background current are measured on every chip as well as three 100% positive internal standards.

The pBDi Control software

pBDi Control is the control and analysis software of the pBDi system. The user monitors and controls the start up and shut down of the device as well as the selection and the progress of methods via pBDi Control. The results of the assay methods are displayed in pBDi Control using a traffic light-based colour code. After identification of a toxin a red traffic light is shown with the warning note that a target molecule was found. Green traffic light indicates that no target substances were found, whereas yellow traffic light reports an error during measurement.

Electrochemical toxin detection

Figure 4: Result analysis with pBDi Control software. As sample a supernatant of S. aureus DSM19044 (strain producing SEB, SEG and SEI) was applied. In pBDi View, register Graph current vs. time course is shown. Buffer flow through the chip chamber (1) was followed by addition of substrate (2). After reaching a plateau of current, flow was stopped and the change in current vs. time was determined for each electrode position (3).

pBDi Toxin Chip 1 & 2

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Figure 3: Layout of pBDi Toxin Chip 1 and pBDi Toxin Chip 2.

Signals of negative control above a critical value or signals of positive control below a critical value can cause invalid measurements. Detailed results can be displayed in pBDi View. In Figure 4 current vs. time course of electrochemical toxin detection is depicted in real time during a measurement. Signals of each electrode are automatically calculated and displayed as real and normalized values in pBDi View. Signals of positive and negative control electrode and corresponding critical values are also indicated (Figure 5). Critical values for evaluation of control electrodes are calculated automatically based on chip lot number. This ensures reproducibility of toxin detection with different chip batches.

Specificity of toxin detection using toxin chips 1 and 2

Specificity of simultaneous toxin detection using Toxin Chip 1 or 2, respectively, is shown in Figure 6. Each target substance produced significant signals only on electrodes immobilized with the respective specific capture antibodies.

No significant cross-reactivity of the antibodies with analyzed target substances was observed.These results demonstrate a good specificity applying toxin concentrations ranging from 2 – 500 ng mL-1.

Beside specificity, sensitivity is of utmost importance for detection of highly toxic bioterrorism relevant toxins in relevant concentration ranges. Limits of detection for proteotoxins are in the pg mL-1 to low ng mL-1 range representing excellent sensitivity for applied short analysis time of 20 minutes. Recently, it was shown, that electrochemical biochip technology was successfully applied in course of a proficiency test on ricin or SEB, respectively, organized in the framework of the EU-project EQuATox [3; 4].

pBDi View signal display

Figure 5: Detailed results of toxin detection. As sample a protein extract from Ricinus communis seeds was applied. For each target sub-stance the critical range, the electrode positions and a bar graph of normalized signals of each electrode is given. Furthermore, the normal-ized and real signal values of the determined slopes are indicated as well as the mean value of the normalized slope of each target. Based on the mean value targets are classified as “negative” or “positive”. Signals of positive and negative control electrodes are also shown.

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For research use only. Not for use in diagnostic procedures.

Bruker DetectionDivision of Bruker Daltonik GmbH

Leipzig · GermanyPhone +49 (341) 2431-30 Fax +49 (341) 2431-404 detection@bruker.comwww.brukerdetection.com

Bruker Detection Corp.

Billerica, MA · USAPhone +1 (978) 663-3660Fax +1 (978) 667-5993 detection@bruker.comwww.brukerdetection.com

References

[1] C.R. Taitt, L.C. Shriver-Lake, M.M. Ngundi, F.S. Ligler, Sensors 8 (2008) 8361-8377[2] E. Nebling, T. Grunwald, J. Albers, P. Schäfer, R. Hintsche, Anal. Chem. 76 (2004) 689-696

[3] S. Worbs, M. Skiba, J. Bender, R. Zeleny, H. Schimmel, W. Luginbühl, B.G. Dorner, Toxins (Basel) 7 (2015) 4987-5010[4] Y. Nia, M. Rodriguez, R. Zeleny, S. Herbin, F. Auvray, U. Fiebig, M.A. Avondet, A. Munoz, J.A. Hennekinne, Toxins (Basel) 8 (9) (2016) E268

Specificity of simultaneous toxin detection

Figure 6: Specificity of toxin detection using pBDi and pBDi Toxin Test Kits. 2 – 500 ng mL-1 toxins were applied as sample. (A) pBDi Toxin Test Kit 1. (B) pBDi Toxin Test Kit 2.

(A) (B)

Conclusion

The pBDi is a portable biothreat agent detection platform useful for automated sensitive and specific detection of biological toxins in approx. 20 minutes. The detection principle is based on ELISA technology in combination with electrochemical signal read out. A decontamination procedure is integrated in the assay ensuring secure operation of pBDi. The pBDi Toxin Chips 1 and 2 allow the parallel detection of BWA relevant toxins such as Botulinum Neurotoxins and Staphylococcal Enterotoxins and even the differentiation of their serotypes.