Received date: April 01, 2016; Accepted date: April 20, 2016; Published date: April 26, 2016
Citation:Bal B, Armstrong PB, Das AP (2016) Development of Indigenous Biosensing Methodology for Rapid and Low Cost Endotoxin Detection System. Sensor Netw Data Commun S1:005. doi: 10.4172/2090-4886.S1-005
Copyright: © 2016 Bal B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Endotoxin is a signature molecule of gram-negative bacteria and is clinically significant as the agent of gramnegative sepsis, a disease condition with high mortality. This study describes an enzyme-substrate reaction using the distilled water lysate of the granular hemocytes (amebocytes) of the mangrove horseshoe crab (Tachypleus gigas), a native of the Bay of Bengal, and a chromogenic peptide which results in the production of yellow spectroscopically monitored product in the presence of endotoxin. The assay is complete within 30 min and shows a lower detection limit of 0.2 EU/mL. The novelty of our approach is the use of mangrove horseshoe crabs are the source of amebocyte lysate instead of the American horseshoe crab (Limuluspoly phemus).The anticipated methodology offers advantages for the South Asian market that include a low cost due to use of indigenous reagents. This method shows exquisite sensitivity and provides a rapid assessment of LPS concentrations. We assume that the commercial development of our assay will reduce the cost of a single chromogenic endotoxin detection test as compared to the cost of present imported kits presently available in Asian and Indian market.
Endotoxin; Detection; Horseshoe crab lysate; Chromogenic; Biosensor
Lipopolysaccharide (LPS, endotoxin), a product of gram negative bacterial infection, induces multiple adverse biological effects including fever [1,2], sepsis [3,4], systemic organ failure , platelet aggregation and thrombocytopenia . Mortality from gram negative sepsis is 30%-50% . Detection and quantification of LPS is important in clinical practice and the identification of LPS in the products of the food processing and pharmaceutical industries is important to ensure quality control and product sterility.The concentration of LPS is usually defined as endotoxin units per millilitre (EU/ml), where1 EU/ml is approximately equal to 0.1 ng/ml. Rapid detection and quantification of endotoxin is indispensable for infection control in biological fluids, medical products, biomedical devices, drugs, recombinant therapeutic products, defence, environment, food and water security [8-10]. The remote exploration satellites destined for solar system targets are monitored for gram-negative bacterial contamination to prevent inadvertent contamination of the target planet . These requirements necessitate the application of highly sensitive bio-sensing methodologies for the detection and quantification of endotoxin at low concentration .
Sepsis is the major cause of death in hospitals with a yearly occurrence of around 18 million worldwide [11,12]. Endotoxininduced sepsis leads to a sum yearly expenditure of $17 billion . Infection and death rates due to gram-negative sepsis have not decreased significantly over the past decade [14,15]. The dangers presented by endotoxin and by gram-negative bacteria demands a rapid and highly sensitive endotoxin detection system to supplant the older slow, laborious, invasive and burdensome techniques [16-18]. The several variants of the Limulus Amebocyte Lysate (LAL) methodology (gel clot, optical, turbidimetric and fluorescent) have proven useful for quantification of the endotoxin content in environmental, food and health care products [19-21] but in South Asia and particularly in India, the LAL test is dependent on foreign suppliers and the costs are too expensive as per the annual report (2012-2013) of National Institute of Biologicals, Ministry of Health and Family Welfare, Govt. of India. Therefore, the development of a low cost, highly sensitive version of this assay for endotoxin detection and quantification for the South Asian market is desirable [22-24].
We report a novel single step chromogenic bio-sensing methodology for a rapid, highly-sensitive and low-cost endotoxin detection system where in a chromogenic assay was investigated as a test for the detection of endotoxin that utilizes the indigenous mangrove horseshoe crab lysate. The endpoint chromogenic method was tested successfully with LPS spiked water samples in different control standard endotoxin concentrations from E. coli O111:B4, ranging from 0.01 EU/mL-50 EU/mL. The method involves a reaction between the native mangrove horseshoe crab lysate and the chromogenic peptide which results in the production of colour in the presence of endotoxin within 36 min. The lower limit of detection obtained was 0.2 EU/mL. The schematic description of chromogenic assay for endotoxin detection is visualised in (Figure 1).
Triethanolamine hydrochloride, Mannitol, Anhydrous calcium chloride, Magnesium chloride hexahydrate was purchased from Himedia for buffer preparation. Standard endotoxin E-ToxateTM (E8029-1vl, SIGMA-ALDRICH) containing 10900 EU/vial and chromogenic substrateBoc-O-benzyl-L-seryl-glycyl-L-arginine-4- nitroanilide hydrochloride (Boc-Ser (Bzl)-Gly-Arg-NH-Np.HCl) was obtained from SIGMA-ALDRICH (USA).
A Scanvac, Labogene lyophilizer was used to lyophilize the amebocyte lysate for long term preservation. Ultra-pure, pyrogen free water was collected from a Milipore Direct Q3 water purification system. Endotoxin calibration was carried out by a microplate method using the 96 well microplate (Tarson 96 Well Flat bottom Micro test plate) in an enzyme-linked immunosorbent assay (ELISA) reader (Mark Microplate reader, Biorad).
Collection of horseshoe crabs
Prior to collection of horseshoe crabs, a short site survey has been carried out to select an opposite nesting site. The Chandipur coast (Latitude - 21.447281 and Longitude - 87.024437) of the Balasore district, Odisha, India was selected due to abundance of these species at this particular location. The crabs were collected with the help of local fisherman and transported in large containers containing sea water to the laboratory [24,25]. Proper care and maintenance was taken during the transport of horseshoe crabs to help reduce any undesired injuries. This method of transport worked well; we have seen no evidence for physical injury to the animals upon their arrival at the laboratory. Upon arrival, the carapace was cleaned of epibionts because this helps to maintain clarity of the sea water of the holding tanks, which is necessary to maintain the health of the captive animals. The entire procedure was conducted with utmost care so as not to damage any horseshoe crab during the process.
Collection of hemolymph
Prior to bleeding, healthy horseshoe crabs were chilled in cold room for 1 h. While holding crab utmost care was taken so as not to hurt animal as it would sensitize the amebocytes leading to rupturing and exocytosis, initiating blood clotting. With the help of other hand cotton soaked in 70% ethanol is used to clean hinge joint. Through hinge joint, a 14 gauze syringe is inserted into the cardiac chamber, up to 1 cm deep as described with a little modification [26,27] . Bleeding was done directly into 15 mL centrifuge tubes containing 0.1 volume of 2% Tween-20 dissolved in 3% NaCl. After the collection of haemolymph horseshoe crabs were returned to their natural habitat that they will be alive. The collected haemolymph was centrifuged at 1000 rpm for 5 min at 4ºC. Care has been taken to centrifuge haemolymph immediately as it decreases the chances of clotting. After centrifugation, each tube has to be scrutinised for any signs of blood clotting in the form haemocytes. Centrifuge tubes containing any signs of blood clotting were discarded. The blood cells were washed repeatedly with LPS - free 3% NaCl solution to remove the proteins of the hemolymph. The wellwashed cell pellet was dissolved in LPS-free distilled water. Insoluble materials were removed by centrifugation, leaving a clear supernatant that is plasma and amebocyte lysate remains at the bottom of the tube shown in Figure 2. The separated lysate was then lyophilized and stored in vials for further analysis.
Chromogenic substrate preparation
The chromogenic substrate Boc-Ser(Bzl)-Gly-Arg-NH-Np. HCl was dissolved in pyrogen free water to make 1 mM of the final stock solution and stored at 4ºC in dark for future use. Imidazoletriethanolamine buffer (0.05M imidazole-triethanolamine, 8% mannitol, 0.02 M calcium chloride, and 0.05 M magnesium chloride pH 8.5) was used for the chromogenic assay and for the endotoxin standardization.
We calibrated the ability of our amebocyte lysate preparation to detect endotoxin in an assay conducted in the wells of a Tarson 96 Well Flat bottom Micro test plate and a microplate reader. Each well received 0.1 mL of diluted endotoxin and 0.1 mL of amebocyte lysate and the preparation was incubated at 37°C for 30 min. Then 0.1 mL of pre-warmed chromogenic substrate, Boc-O-benzyl-L-seryl-glycyl- L-arginine-4-nitroanilide hydrochloride was added into each well and the preparation was incubated at 37°C for another 6 min. The reaction was stopped by adding 0.1 mL of 25% glacial acetic acid into each well and the concentration of chromogenic product was determined with an ELISA reader at an absorbance of 405 nm. Endotoxin concentration in 0.1ml diluted urine samples collected from six different patients of the SUM hospital, Bhubaneswar, Odisha, India were determined using the same endotoxin calibration method as described above.
The proposed methodology involves a reaction between the proteases of an endotoxin-activated protease cascade of the distilled water lysate of the granular amebocyte blood cells of the horseshoe crab and a chromogenic substrate for those proteases. Formation of the extracellular blood clot of the horseshoe crab is initiated by the exocytotic secretion of the structural protein of the clot, coagulogen, and its proteolytic modification by the members of a protease cascade that is initiated by the auto-catalytic activation of the initial protease of the cascade, factor C. Factor C activates a second protease; factor B, which activates the third protease in this cascade, proclotting enzyme , which administers two proteolytic cleavages C-terminal to Gly- Arg moieties in molecules of the soluble zymogen, coagulogen. This promotes the polymerization of coagulin into the insoluble fibres of the extracellular blood clot . Factor C, the initiator of this proteolytic cascade, is activated by its binding of endotoxin (a.k.a., lipopolysaccharide, LPS), which provokes a structural rearrangement of the pro-factor C molecule, allowing its proteolytic auto activation [21,30]. Our quantification of endotoxin concentrations depends on the assay rate of the proteolytic cleavage of the substrate, Gly-Arg-pnitroanilide by these three proteases. The p-nitroanilide leaving group of the substrate is assayed spectrophotometrically at 410 nm.
Endotoxin detection in clinical samples with the chromogenic horseshoe crab lysate reagent
The endotoxin and amebocyte lysate reagent were incubated for 36 min and the concentrations of the proteolytically generated chromogenic substance were determined using a microplate reader. A standard curve for endotoxin detection showed a dynamic range for the assay from 0.1 EU/ml-1.2 EU/ml Figure 3. The urine samples were analysed for presence of endotoxin and out of six urine samples four samples were found to be positive and two samples were negative. Sample S5 and S4 had higher endotoxin of about 0.793 EU/mL and 0.754 EU/mL, whereas S1 and S3 were 0.486 EU/mL and 0.154 EU/mL respectively (Figure 4). As the clinical endotoxin permissible limit is below 0.2 EU/mL  therefore sample S5 and S4 was much infected sample as compare to S1 and S3 as shown in Figure 4. Sample S2 and S6 were found to be negative.
Estimated cost analysis of the developed technique
The principal benefit of our product for the South Asian market is the local production of amebocyte lysate from locally collected mangrove horseshoe crabs, rather than using lysate prepared in the United States or China. At present the cost of LIMUSATE@ LAL AND PYROSTAR LAL- Kits being imported from United States is around 50,000 INR, and the Kinetic-QCL 192 Test Kit from Lonza costs around 58,000 INR data collected from list of import records released by government of India for the keyword `Vials` under Harmonized System (HS) code 38220090 and received budgetary quotations from these commercial manufactures. Hence a locally produced lysate will be less costly to develop a cheaper endotoxin detection methodology.
Abundance of the mangrove horseshoe crab in the Bay of Bengal
A problem that at present is difficult to evaluate is the extent to which the local populations of mangrove horseshoe crabs in the Bay of Bengal can support the large scale production of amebocyte lysate. The three pharmaceutical companies in the United States that produce amebocyte lysate using the blood of L. polyphemus bleed an estimated 250,000 animals per year  with a mortality of 15%-20%  and admittedly, anthropogenic activity has compromised some of the nesting sites of the mangrove horseshoe crab along the coast of the Bay of Bengal for both Carcinoscorpius rotundicauda and Tachypleus gigas . But it can be hoped that the use of the mangrove horseshoe crab for the production of the high-value amebocyte lysate will be coupled with the imposition of the appropriate conservation measures that will support the health of the population of mangrove horseshoe crabs, which is necessary for the continuance of this important industry. We envision the establishment of an amebocyte lysate industry as an impetus for the conservation of its essential resource, the mangrove horseshoe crab.
This report describes an endotoxin monitoring system that uses reagents indigenous to India and which is based on the endotoxin-based activation of serine proteases in mangrove horseshoe crab amebocyte lysate assay and on the assay of protease activity with a chromogenic peptide substrate. These chromogenic assays using the mangrove horseshoe crab lysate can determine endotoxin with sensitivity of 0.02 EU/mL within 36 min. The proposed methodology exhibits numerous advantages such as a short detection time and exquisite sensitivity. This method does not require complex and costly devices and can be quantified with standard ELISA reader. Additionally the use of a locally produced lysate from the mangrove horseshoe crab will be less costly to develop an inexpensive endotoxin kit. The proposed method requires only small volumes of the test sample. This method performed well for the detection and quantification of concentrations of endotoxin in human clinical urine samples.
The authors would like to acknowledge the Department of Science and Technology (DST) [IDP/MED/2013/02], Government of India for providing financial support for this research.
The authors declare no conflict of interest.