Microchip-based express diagnostics of botulinum toxins
Botulism (from lat. botulus – sausage) is heavy toxic-infectious disease characterized by lesions of the nervous system (mostly oblongata and spinal cord). The source of infection can be food, water, aerosols containing botulinum toxin, which is produced by spore-forming bacteria Clostridium botulinum. Despite the fact that cases of botulism are logged less frequently than other intestinal infections and poisoning, this disease continues to be dangerous to human life [1-4].
Seven types of the pathogen are known – A, B, C (subtypes C1 and C2), D, E, F and G, which differ in antigenic structure of secreted exotoxin . Outbreaks of botulism is most often caused by type A of toxin, less – by types B, C, E and F. Toxin D causes diseases only in animals and waterfowl .
The symptoms of botulism appear suddenly – dry mouth, muscle weakness, impaired vision, difficulty swallowing, change of pitch and timbre of voice, rapid breathing. The disease develops rapidly and may last from one to two months. Deaths occur mainly due to respiratory failure. The incubation period elapses from a few hours up to 2-5 days, averaging – 18-24 hours.
Methods of diagnosis
Currently used methods of diagnosis of botulism are characterized by a duration of analysis from 1 to 10 days, which is at least two times longer than the incubation period of the disease. Material for bacteriological examination are the feces and vomit of the patient, the gastric washings, the contents of wounds (wound botulism), suspected food .
The following methods of diagnostics of toxin of botulism are known :
clinical – observation of infected animals (white mice, guinea pigs, rabbits), analysis of blood, hyperimmunization;
bacteriological – seeding of Cl.botulinum on MPA with 2% glucose in Kitt-Tarozzi medium, Hottinger broth with 1% glucose, isolation of pure cultures, the receipt and accumulation of the bacterial mass and produced toxin;
cultural-morphological and biochemical – study of sacharolytic and proteolytic properties of strains of Cl.botulinum;
microscopic – light and fluorescence microscopy;
serological – neutralization test, indirect haemagglutination, the enzyme-linked immunosorbent assay;
radiobiological – gamma radiation inactivation of Cl.Botulinum;
chemical – inactivation of Cl.botulinum and converting toxins to toxoids with 40% formalin solution;
microchip-based diagnostics .
The most modern method of diagnosis of botulism is based on the use of devices with a special microchip. In particular, this principle is implemented in universal portable analyzer ePaTOX II, which is designed for highly sensitive detection of proteins, toxins, nucleic acids and other biomolecules in samples of different species. The device allows to detect the botulinum toxin of types A, B, E and F, and can be used in hospital laboratories and in mobile detection systems .
The principle of microchip-detection with the ePaTOX II is based on an electrochemical reaction. The process utilizes chips with extremely fine, nanoscale electrode structures produced using the state of the art silicon semiconductor technology. A biochip of this kind includes 16 measuring positions which are then loaded with various receptor molecules specific to the application. Depending on how the biochip is configured, this allows scientists to detect multiple toxins or pathogens in parallel on a single chip. The sample to be studied is automatically pumped across the chip during the detection process, allowing the target molecules in the sample to bind to their complementary receptor molecules (e.g., antibodies, oligonucleotides), which are immobilized on the surface of the chip. This step is followed by enzyme marking.
Fixed on the chip in this way, the enzyme then converts a substrate that can be detected in an electrochemical reaction. The electrode structures on the chip make it possible to measure an electrical signal – the size of the signal is directly proportional to the concentration of the target molecules in the sample. This electrochemical detection principle makes it possible to achieve high analytical sensitivity, and the resulting detection system is immune to the effects of turbidity and other sources of optical interference. Full detection of botulinum toxin typically takes about 20 minutes.
Operators are able to use the instrument after only a brief introduction thanks to uncomplicated, ready-to-use kits and user-friendly control/analysis software.
Thus, the modern innovative method of diagnosis of botulism by means of the analyzer of toxins and pathogens on the basis of a microchip allows to reduce considerably time of the analysis and in due time to start treatment of an infection, having facilitated the course of a disease. It is also possible prevention by analysis of food for the presence of botulinum toxin, that gives an opportunity to prevent disease. ■