WO2021053691A1

WO2021053691A1 - Device and assay for detection of antibiotics in industrial effluents

Info

Publication number: WO2021053691A1
Application number: PCT/IN2020/050795
Authority: WO
Inventors: Sreya KRISHNAKUMAR; Swaroop MHASAWADE; Navya SETHU; Chandrakant TAGAD; Renu VYAS
Original assignee: Mit School Of Bioengineering Sciences & Research
Priority date: 2019-09-16
Filing date: 2020-09-16
Publication date: 2021-03-25

Abstract

Disclosed are a device (150) and an assay for detection of antibiotics in industrial effluents. The reaction mechanism used in the device (150) is sensitive and specific towards aminoglycoside antibiotics and requires minimal sample preprocessing. The detection mechanism used in the device (150) is cell-free, enzyme free and hence has good reproducibility and does not require ancillary equipment. The use of automated syringe pumps ensures precision in reagent additions, thereby minimizing human error to a great extent. The assay of the present invention utilizes fatty acid peroxidation as a means for antibiotic quantification.

Description

DEVICE AND ASSAY FOR DETECTION OF ANTIBIOTICS IN INDUSTRIAL EFFLUENTS

Field of the invention:

The present invention relates to the field of sensing devices and methods for antibiotic detection and particularly, to a device and an assay for detection of antibiotics in industrial effluents. More particularly, the present invention relates to a miniaturized, cost efficient fiber optic sensing device and a cost effective, rapid and sensitive assay developed for the detection of aminoglycoside based antibiotics such as gentamicin.

Background of the invention: Antibiotics are widely being used for treating diseases and promoting animal growth worldwide. Antibiotics are being extensively applied to human and veterinary medicine due to their capability to enhance growth rates and improve feed efficiency. Widespread use of antibiotics for the treatment of food-producing animals generates risk to human health due to transmission of residues and metabolites of these antibiotics into the food chain. Undigested antibiotics are excreted as such, resulting in increasing level of the antibiotics in water bodies creating a serious threat to aquatic life as well as human health. Environmental pollution due to antibiotics is a major cause of concern due to their unmediated consumption and disposal, which, left undetected, leads to antibiotic resistance in microorganisms that come in contact with antibiotics.

Amongst the various classes of antibiotics, aminoglycoside antibiotics have severe side effects like nephrotoxicity and ototoxicity. Thus, the presence of aminoglycosides in animal-origin food can be a serious threat to human health. Owing to this, the European Agency for the Evaluation of Medical Products (EMEA/MRL/803/01-FINAL, 2001) and the Ministry of Agriculture of China (Official Journal, 2002) have established maximum residue limits (MRLs) for edible tissues and milk for consumer protection.

Numerous efforts have been made to develop analytical methods for qualitative/quantitative determination of antibiotics in the last two decades. Various sophisticated analytical techniques like gas chromatography combined with mass spectrometry (GC-MS), gas chromatography coupled with electron capture (GC-EC), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), diode array (DA), flame ionization (FI) and enzyme-linked immunosorbent assay (ELISA) are being used for the detection of antibiotics with accuracy and precision.

However, these techniques have limitations such as high cost, time-consuming process of analysis, and requirement of trained personnel. Therefore, there is a growing demand for sensitive, selective, robust, and rapid sensing devices for detection of antibiotic residues for human health and safety.

There are very few reports on portable sensing techniques which can be effectively used for the detection of aminoglycosides for real-time applications. Hence, a simple, cost-effective and miniaturized sensing device for the detection of aminoglycosides needs to be developed.

US patent 4330621 elucidates a method to detect aminoglycoside antibiotics in blood by rendering the -SH groups in the medium insoluble, extracting them, and detecting thiol in the remaining solution spectrophotometrically. This was followed by treatment with an aminoglycoside- specific enzyme and spectrophotometric detection. The differences between the optical densities were used as a measure of antibiotic quantification. However, the performance of the method mentioned is not convenient for samples excluding blood, because (a) enzyme activities depend on local environment to a great extent, and hence requires strict maintenance of physiological conditions, (b) the usage of spectrophotometry as a detection method is inconvenient and expensive for real time detection and monitoring.

US4239745A relates to a sensitive method for antibiotic detection in body fluids, dairy and food industry. But its scope is limited to a lab technology, where sophisticated equipments are necessary. Also, microorganisms which are supersensitive to the antibiotic have to be used for high sensitivity, which is a time-consuming process.

Another US patent 8476064 describes methods including microbial growth inhibition and antibody-mediated reactions as a detection strategy. In real time applications, specifically for environmental samples, some methods are (a) not specific to antibiotics and may result in false positives (b) time and resource consuming, requires heavy apparatus and strict environmental conditions.

Reference may be made to US patent number US8895260 that discloses a strategy for detecting ribosomal antibiotics using a comparative analysis of bacterial strains containing bacterial, chimeric mitochondrial and chimeric cytosolic bacterial ribosomes. These methods are also heavy on cost and high precision technique, and are not suitable for testing of real time environmental samples without extensive sample pre-processing.

US4073694 reveals a method of determining the concentration of an aminoglycoside antibiotic in blood tested with gentamicin, amikacin, tobramycin using a microbiological assay. However, the method takes a long time of nearly two hours for detection, which cannot be used in a portable device. As its response time is too long, its application has limited scope in laboratory analysis only.

Yet another US6749740 claims an invention for a small volume sensor based on current and time required to electrolyze an analyte using a known amount of charge in known time duration. Since the method is based on the redox potential of the analyte in the sample, it is not sensitive for samples which may add to the charge of the analyte. Such a system is difficult to implement for samples with unknown constitutions.

Furthermore, US4101383 discloses an optical system for the measurement of change in absorbance of microbes in a culture broth on treatment with antibiotics. Large sample and culture volumes and low sensitivity restrict its potential for quantitative estimation in effluents.

To regulate the antibiotic content in effluents, especially in the industrial scenario, a specific detection apparatus is required. Thus, in order to overcome the liabilities of the abovementioned prior art, there is a need for the design of a self- contained device that is more sensitive and specific to the analyte, more tolerant to interferences, has a rapid response time, is portable, affordable and cost-effective.

Objects of the invention:

An object of present invention is to develop a simple, cost-effective and miniaturized sensing device for detection of antibiotics such as aminoglycosides.

Another object of present invention is to provide an assay for detecting and quantifying antibiotic content in industrial effluents for regulation purposes.

Summary of the invention:

Accordingly, the present invention provides a device for detection of antibiotics in industrial effluents. The device comprises a casing, a display, a switch, a mould, an optical sensing mechanism, a base, an electronic circuitry, a battery, a calibration switch, a measure switch and a motor driver circuitry. The casing encloses the mould, the optical sensing mechanism, the base, the electronic circuitry and the battery therein. The mould is mounted on the base. Specifically, the mould and the base are made of Polydimethylsiloxane material. The mould includes a plurality of syringes, a reaction well and a plurality of micro channels. The plurality of syringes is used for storing reagents used in an assay for detection of the antibiotics. The sample to be assayed/ analyzed is loaded in the reaction well and the reaction mixture is drained out through a drain channel. The reaction well is connected to the plurality of syringes through the plurality of micro channels using a timer based pumping mechanism. The timer based pumping mechanism enables automatic loading of accurate amount of reagents from the plurality of syringes to the reaction well. The optical sensing mechanism is positioned on the base. The optical sensing mechanism includes a light source, a first optical fiber cable, a second optical fiber cable and a photo detector. The light source is connected to the reaction well through the first optical fiber cable. Specifically, the light source is a light-emitting diode. The light from the light source is emitted into the reaction well. The photo detector is positioned in line with the light source. The photo detector is connected to the reaction well through the second optical fiber cable.

The electronic circuitry includes an amplifier and a microcontroller. The microcontroller is connected to the mould and programmed to control the timer based pumping mechanism with the help of the motor driver circuitry. The battery is used to provide power to the microcontroller. The switch is positioned on a side of the device and used for operation thereof. The display is provided on the casing to show a concentration of the antibiotic present in the sample. Specifically, the display is organic light emitting diodes display. The calibration switch is used for calibration of the device and the measure switch is used for taking the measurement with the sample.

In another aspect, the present invention provides a cost effective, rapid and sensitive assay for detection of aminoglycoside based antibiotics such as gentamicin.

Brief description of the drawings:

The objects and advantages of the present invention will become apparent when the disclosure is read in conjunction with the following figures, wherein Figure 1 shows a device for detection of antibiotics in industrial effluents, in accordance with the present invention;

Figure 2 shows a schematic diagram of the device for the detection of the antibiotics in the industrial effluents, in accordance with the present invention;

Figure 3 shows a top view of the device of figure 1, in accordance with the present invention;

Figure 4 shows a lateral view of internal components of the device of figure 1, in accordance with the present invention; and

Figure 5 shows a top view of the internal components of the device of figure 1, in accordance with the present invention.

Detailed description of the invention:

The foregoing objects of the invention are accomplished and the problems and shortcomings associated with the prior art techniques and approaches are overcome by the present invention as described below in the preferred embodiment.

In one aspect, the present invention provides a new miniaturized, cost efficient fiber optic sensing device that is developed for the detection of the antibiotics. In another aspect, the present invention provides a cost effective, rapid and sensitive assay for detection of aminoglycoside based antibiotics such as gentamicin. The assay is based on the iron dependent peroxidation of free fatty acids by gentamicin.

This present invention is illustrated with reference to the accompanying drawings, throughout which reference numbers indicate corresponding parts in the various figures. These reference numbers are shown in bracket in the following description below.

Referring to figures 1 to 5, in one aspect, a device (150) for detection of the antibiotics in industrial effluents, in accordance with the present invention is shown. The device (150) comprises a casing (10), a display (20), a switch (30), a mould (40), an optical sensing mechanism (not numbered), a base (60), an electronic circuitry (70), a battery (80), a calibration switch (90), a measure switch (100) and a motor driver circuitry (110).

All the components of the device (150) including the mould (40), the optical sensing mechanism, the base (50), the electronic circuitry (70) and the battery (80) are enclosed in the casing (10). The display (20) is provided on the casing (10) to show the concentration of the antibiotic present in a sample. In an embodiment, the display (20) is organic light emitting diodes (OLED) display. The switch (30) is positioned on a side of the device (150) and used for the operation thereof. The calibration switch (90) is used for calibration of the device (150) and the measure switch (100) is used for taking the measurement with the sample. The mould (40) along with the optical sensing mechanism is mounted on the base (60). In an embodiment, the base (60) is made of Polydimethylsiloxane (PDMS) material.

The mould (40) comprises a plurality of syringes (32) (hereinafter referred as, “the syringes (32)”), a plurality of micro channels (34) (hereinafter referred as, “the micro channels (34)”) and a reaction well (36). The reagents used in the assay are stored in the syringes (32) and are delivered to the reaction well (36) as per the assay protocol. The syringes (32) are connected to the reaction well (36) by the micro channels (34) using a timer based pumping mechanism achieved by a microcontroller (70) and the motor driver circuitry (110). The timer based pumping mechanism enables automatic loading of accurate amount of reagents from the syringes (32) to the reaction well (36) in accordance with the assay protocol. Also, the pumping action exhibits unidirectional flow preventing any backflow of reagents. The sample to be assayed/analyzed is loaded in the reaction well (36) and the reaction mixture is drained out through a drain channel (38). In an embodiment, the mould (40) is made of Polydimethylsiloxane (PDMS) material which enables the reagents (fluids) to flow completely without sticking on edges of the mould (40), thus minimizing contamination issues. In a preferred embodiment, the mould (40) comprises four syringes (32) and four micro channels (34). However, it is understood here that number of the syringes (32) and the micro channels (34) used in the device (150) may vary in other alternative embodiments of the present invention as per the intended application.

The optical sensing mechanism includes a light source (42), a first optical fiber cable (44), a second optical fiber cable (46) and a photo detector (48). In an embodiment, the light source (42) is a light-emitting diode (LED). The light source (42) is connected to the reaction well (36) through the first optical fiber cable (44). The light from the light source (42) is emitted into the reaction well (36). The photo detector (48) is positioned in line with the light source (42). The photo detector (48) is connected to the reaction well (36) through the second optical fiber cable (46). In a preferred embodiment, the light source (42) and the photo detector (48) are selected based on spectral properties of the reaction mixture in the reaction well (36).

The electronic circuitry (70) includes the microcontroller (62) and an amplifier (not shown). The microcontroller (62) is connected to the mould (40) and is programmed to control the timer based pumping mechanism with the help of the motor driver circuitry (110). The device (150) also sends the amplified output voltage to the microcontroller (62) that in turn is connected to the display (20). The microcontroller (62) is powered with the battery (80) through the switch (30).

In another aspect, the present invention provides a novel assay for the detection of the antibiotics in the industrial effluents. Specifically, the assay is developed for the detection of aminoglycoside based antibiotics in a sample. The assay of the present invention utilizes fatty acid peroxidation as a means for antibiotic quantification.

In a first step, the sample to be analyzed along with the reagents is loaded from the syringes (32) in the reaction well (36). For example, for the detection of the aminoglycoside antibiotic, the assay is performed by adding an aqueous sample containing the aminoglycoside antibiotic into an aqueous solution of polyunsaturated fatty acid. The reaction is accelerated by the addition of a ferrous catalyst, and the subsequent product formed is quantified by the addition of a chromogenic dye. For the aminoglycoside antibiotic, the reagents include, but not limited to, polyunsaturated fatty acids such as arachidonic acid and linoleic acid. The final concentration of the polyunsaturated fatty acids present in the assay is in the range of 100 ng/ml to 1 pg/ml. The ionic catalysts such as ferrous salts having final molar concentration in the assay in the range of range 1 mM -10 mM, and free radical sensitive chromogenic dyes such as tetramethylbenzidine and nitro blue tetrazolium in the range of 100 ng/ml to 1 pg/ml are utilized. However, it is understood here that the reagents, the catalysts and the dyes used and concentration thereof may vary in other alternative embodiments of the present invention as per the type of the antibiotic to be assayed.

In next step, the maximum intensity of the light is passed by the light source (42) through the first optical fiber cable (44) to the reaction well (36). Thereafter, the transmitted light intensity is measured in terms of voltage using the photo detector (48) which varies from 25 mV - 1360 mV. In next step, the transmitted light intensity through the colored product is detected using the optical sensing mechanism. In a final step, the respective concentration of the antibiotic present in the sample is displayed on the display (20) of the device (150).

The invention is further illustrated hereinafter by means of examples.

Examples:

Assay for quantification of gentamicin:

The assay for quantification of gentamicin was developed based on iron dependent peroxidation of arachidonic acid by gentamicin. Polyunsaturated fatty acids such as arachidonic acid present in the sample were oxidized in the presence of ferrous sulfate (FeSO and gentamicin to release free radicals. These free radicals oxidized the transparent tetramethylbenzidine (TMB) to blue- green oxTMB. The intensity of blue-green color produced was proportional to the amount of gentamicin in the sample.

The color intensity of the assay product increased above a baseline value as a function of the concentration of gentamicin. Transmitted light intensity through the colored product was detected using the optical sensing mechanism and displayed on the OLED (20) of the device (150).

The synthetic reaction scheme developed for a colorimetric assay for the detection of gentamicin using the device (150) is shown below. However, it is understood here that the similar mechanism can be applied to dyes which respond to fatty acid peroxidation including, but not limited to, transparent tetramethylbenzidine (TMB).

Advantages of the invention: 1. The assay of the present invention utilizes fatty acid peroxidation as a means for antibiotic quantification. This is the first known instance of the use of fatty acid peroxidation as a means for antibiotic quantification combined with free radical estimation using dyes such as, but not limited to tetramethylbenzidine and nitro blue tetrazolium. 2. The reaction mechanism used in the device (150) is sensitive and specific towards aminoglycoside antibiotics and requires minimal sample preprocessing. 3. The detection mechanism used in the device (150) is cell-free, enzyme free and hence has good reproducibility and does not require ancillary equipment.

4. The optic detection system used in the device (150) is simple to construct, use, maintain and calibrate. The detection system is customizable and calibrated for different assays. By changing the reagents in the respective chambers, the device (150) is used for any class of antibiotics.

5. The use of automated syringe pumps ensures precision in reagent additions, thereby minimizing human error to a great extent.

6. The device (150) does not absorb or bind to liquid entities during reaction, is easy to clean and operable in extremely low reaction volumes.

7. The device (150) can be portable and a direct readout of antibiotic concentration is obtained which makes it useful in in-situ applications.

8. The detection time of the device (150) and the assay of the present invention are limited to a very few minutes.

9. The sensitivity of the device (150) can further be increased by addition of amplification circuits with more gain.

10. The device (150) can be calibrated for each use by pressing a button, which can also be achieved by a plurality of wells.

11. Due to the timer based pumping mechanism of the reagents, it is not compulsory that user should have technical knowledge on that device (150).

12. The device (150) facilitates applications in various industries such as pharmaceuticals, textile, animal feed, fisheries, water treatment plants, and the like, provided sample preprocessing is already performed.

13. As the device (150) is a programmable one, it can be reprogrammed for using in the detection of other antibiotic classes according to their limits of detection (LOD).

14. Since the optical sensing mechanism is used in the device (150), there are no mechanical interferences, which affect the functioning of the optical sensing mechanism, and therefore, less maintenance has to be provided. The foregoing objects of the invention are accomplished and the problems and shortcomings associated with prior art techniques and approaches are overcome by the present invention described in the present embodiment. Detailed descriptions of the preferred embodiment are provided herein; however, it is to be understood that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in virtually any appropriately detailed system, structure, or matter. The embodiments of the invention as described above and the methods disclosed herein will suggest further modification and alterations to those skilled in the art. Such further modifications and alterations may be made without departing from the scope of the invention.

Claims

We claim:

1. A device (150) for detection of antibiotics in industrial effluents, the device (150) comprising: a mould (40) mounted on a base (60), the mould (40) having, a plurality of syringes (32) for storing reagents used in an assay for detection of the antibiotics, and a reaction well (36) connected to the plurality of syringes (32) through a plurality of micro channels (34) using a timer based pumping mechanism, a sample to be assayed being loaded in the reaction well (36) and the reaction mixture is drained out through a drain channel (38); an optical sensing mechanism positioned on the base (60), the optical sensing mechanism having, a light source (42) connected to the reaction well (36) through a first optical fiber cable (44), the light from the light source (42) being emitted into the reaction well (36), and a photo detector (48) positioned in line with the light source (42), the photo detector (48) connected to the reaction well (36) through a second optical fiber cable (46); an electronic circuitry (70) having an amplifier and a microcontroller (62) connected to the mould (40), the microcontroller (62) being programmed to control the timer based pumping mechanism with the help of a motor driver circuitry (110); a battery (80) for powering the microcontroller (62); a switch (30) positioned on a side thereof; a casing enclosing the mould (40), the optical sensing mechanism, the base (50), the electronic circuitry (70) and the battery (80) therein; a display (20) provided on the casing (10) to show a concentration of the antibiotic present in the sample; a calibration switch (90) for calibration thereof; and a measure switch (100) for taking the measurement with the sample.

2. The device (150) as claimed in claim 1, wherein the mould (40) and base (60) are made of Polydimethylsiloxane material.

3. The device (150) as claimed in claim 1, wherein the light source (42) is a light-emitting diode.

4. The device (150) as claimed in claim 1, wherein the display (20) is organic light emitting diodes display.

5. The device (150) as claimed in claim 1, wherein the timer based pumping mechanism enables automatic loading of accurate amount of reagents from the plurality of syringes (32) to the reaction well (36).

6. An assay for detection of antibiotics in industrial effluents, the assay being carried out using a device (150), the assay comprising: loading a sample to be analyzed along with reagents from a plurality of syringes (32) in a reaction well (36) of the device (150); passing maximum intensity of light by a light source (42) through a first optical fiber cable (44) to the reaction well (36); measuring transmitted light intensity in terms of voltage using a photo detector (48); detecting the transmitted light intensity through a colored product using an optical sensing mechanism; and displaying concentration of the antibiotic present in the sample on a display (20) of the device (150).

7. The assay as claimed in claim 6, wherein the antibiotics are aminoglycoside based antibiotics.

8. The assay as claimed in claim 7, wherein the reagents used are polyunsaturated fatty acids such as arachidonic acid and linoleic acid, ionic catalysts such as ferrous salts, and free radical sensitive chromogenic dyes such as tetramethylbenzidine and nitro blue tetrazolium.

9. The assay as claimed in claim 8, wherein tetramethylbenzidine and nitro blue tetrazolium are used in the range of 100 ng/ml to 1 pg/ml.

10. The assay as claimed in claim 6, wherein the light source (42) is a light- emitting diode and the display (20) is organic light emitting diodes display.