Fabrication of microfluidic device for Aflatoxin M1 detection in milk samples with specific aptamers

Abstract

This study describes the colorimetric detection of aflatoxin M1 (Afl M1) in milk samples using a microfluidic paper-based analytical device (µPAD). Fabrication of µPADs was done using a simple and quick approach. Each μPAD contained a detection zone and a sample zone interconnected by microchannels. The colorimetric assay was developed using unmodified AuNPs as a probe and 21-mer aptamer as a recognition molecule. The free aptamers were adsorbed onto the surface of AuNPs in absence of Afl M1, even at high salt concentrations. The salt induced aggregation of specific aptamers occurred in presence of Afl M1. Under optimum conditions, the analytical linear range was found to be 1 µM to 1 pM with limit of detection 3 pM and 10 nM in standard buffer and spiked milk samples respectively. The proposed aptamer based colorimetric assay was repeatable, quick, selective, and can be used for on-site detection of other toxins in milk and meat samples. Mycotoxins are secondary metabolites produced by filamentous fungi belonging to the genera Aspergillus and Penicillium 1. Mycotoxins are also found in animal derived foods such as milk due to intake of contaminated feed 2,3. Considering agricultural and economical aspects and possible implications on public health, the most relevant mycotoxins are aflatoxins, ochratoxins, fuminosins, T-2 toxin, and zearalenone (ZEA) 4. Aflatoxins pose a huge economic burden as they cause around 25% or more loss of the world's food crops every year 5. Afl M1 (4-hydroxy aflatoxin B1) and M2 (4-dihydroxy aflatoxin B1) have well proven carcinogenic and mutagenic potentiality and pose severe health consequences on milk consumers, including the risk of cancer and stunted growth in children below the age of 5 years 6. When B1 is ingested by a cow, it is secreted as hydroxylated metabolite aflatoxin M1 (Afl M1) in the urine and milk of the cow 7. Consumption of food containing aflatoxin concentrations of one milligram/kilogram or higher has been suspected to cause aflatoxicosis 8 , the prognosis of which consists of acute liver failure, jaundice, lethargy, and nausea, eventually leading to death within 1 to 2 weeks, based on past reports 9. Thus there is a need to develop rapid low cost technology based highly specific methods for detection of aflatoxins to improve surveillance and control in rural areas. A plethora of analytical techniques are available for aflatoxin M1 detection, ranging from chromatography and HPLC-MS used for regulatory control in official laboratories to rapid test kits for grain silos and farmers, especially for surveys when outbreaks occur 10,11. Conventional techniques for the detection of aflatoxins include gas chromatography (GC), fluorescence or UV based detection, thin layer chromatography (TLC) and enzyme linked immunosorbent assay (ELISA) 6-12. These techniques are routinely used and yield reliable results, however they are expensive, time consuming, require large scale instrumentation and large amounts of hazardous chemical reagents. Advancement in the development of bios...

Graphene based sensors

“…Biosensors are classified based on the different type of recognition element, transducer or amplifier used. On the basis of the type of recognition element [12] , biosensors are often classified into enzymatic sensors [14] , [15] , [16] , immunosensors [13] , [17] , [18] , [19] , DNA based sensors [20] , [21] , [22] , aptamer based sensors [23] , [24] or whole cell microbial biosensors [25] , [26] , [27] , [28] . Based on the type of transducer, biosensors can be categorized into four types- Electrochemical biosensors [29] , [30] , Optical biosensors [31] , [32] , [33] , Piezoelectric biosensors [34] , [35] , [36] , and Thermal biosensors [37] , [38] , [39] .…”

Recent Advances in Microfluidic Devices for Contamination Detection and Quality Inspection of Milk

“…1881, 2006) [ 1 ]. The recent literature thus contains many microfluidic lab-on-a-chip and lab-on-paper devices for the detection of AFB1 in milk [ 53 , 54 , 55 ] and AFM1 [ 56 , 57 , 58 , 59 ] in milk samples. For example, Sheini [ 54 ] developed a microfluidic lab-on-paper colorimetric aggregation assay sensor array for the detection of five mycotoxins classified into three categories, namely aflatoxins, ochratoxins A (OTA), and zearalenone.…”

Using Aptamers as a Novel Method for Determining GnRH/LH Pulsatility

“…Aptamers can be modified (more readily than antibodies) to increase their specificity, ligand affinity, half-lives and/or facilitate their incorporation into sensor devices [ 56 , 57 ]. Recently described aptamer-based devices include a lateral flow strip for the detection of hepatitis C core antigen in serum (with a colour-change produced by a positive result visible to the naked eye within 10 min) [ 58 ], a microfluidic paper device for detection of aflatoxin B1 in milk [ 59 ] and standard blood glucose meters have been adapted for quantification of interferon-γ [ 60 ] and cocaine in human serum [ 61 ] using aptamers. Additionally, aptamer-based devices have also been used for in vivo measurement of cocaine in different regions of rat brains using modified electrodes [ 62 ] and for in vivo measurement of antibiotics in ambulatory rats using implanted sensors [ 63 ].…”