MIP-based protein profiling: A method for interspecies discrimination

Abstract Due to recent public concern and interest in the authenticity and origin of meat, for example, the 2013 “horsemeat scandal” in the human food chain, novel sensor strategies for the discrimination between protein species are highly sought after. In this work, molecularly imprinted polymers (MIPs) are utilised for protein discrimination using electrochemical sensor and spectrophotometric techniques. MIP selectivity between two proteins of similar molecular weight (haemoglobin and serum albumin) were compared across three different species, namely pork, beef and human. Bulk MIPs resulted in Kd and Bmax values of 184 ± 23 μM, and 582 μmol g−1 for BHb, 246.3 ± 26 μM, and 673 μmol g−1 for HHb; 276 ± 31 μM, and 467 μmol g−1 for PHb. With the aid of chemometrics, i.e. multivariate analysis and pattern recognition, distinctive protein profiles have been achieved for species discrimination in both spectrophotometric and electrochemical analysis experiments. MIP suitability and selectivity within complex matrices was also assessed using urine, human plasma and human serum. Pattern recognition MIP-based protein profiling demonstrated positive outputs yielding either a ‘bovine’ or ‘not-bovine’ outcome (p = 0.0005) for biological samples spiked with/without bovine using respective bovine haemoglobin MIPs.


29
Proteins are essential parts of organisms and participate in virtually every process within 30 cells 1

67
There has been recent public concern and interest in the authenticity and origin of meat in 68 the human food chain. For example, the 2013 'horsemeat scandal' were the Food Safety

69
Authority of Ireland (FSAI) announced the discovery of horse DNA in supposedly 100% beef 70 burgers sold in British and Irish supermarkets 19 . In light of this, novel sensor strategies for 71 the discrimination between protein species are highly sort out. Recent developments using 72 60 MHz 1 H NMR as a screening tool for distinguishing beef from horse meat has been 73 demonstrated 20 . While this represents a feasible high-throughput approach for screening 74 raw meat, the method is inherently not portable and so cannot be used in-field. In this work, 75 we look to discriminate between key proteins in 3 species using cheap, portable and 76 synthetic smart material MIPs. MIP selectivity for two proteins of similar molecular weight 77 (haemoglobin and serum albumin) are compared across three different species, namely 78 Porcine (pig), Bovine (cow) and human using the combined latter mentioned techniques.

79
Haemoglobin (Hb) is a well-known allosteric protein for its carbon dioxide and oxygen 80 transport in the blood, as well as regulating blood pH 21 . Hb is approximately 64.5 kDa in size 81 (~5 nm) and has an iso-electric point (pI) of 6.8. Compared to smaller proteins, Hb will 82 possess more anchor points with functional monomers and hence more flexible conformational transitions in the imprinting process 21 . This results in more difficulties for Hb 84 to form imprinted sites. Serum albumin (SA) with a molecular weight of 66.4 kDa and a pI of 85 4.7, is the main monomeric globular protein of plasma, and has a good binding capacity for 86 water, Ca 2+ , Na + , K + , fatty acids, hormones, bilirubin and drugs. SA, particularly from bovine 87 (BSA), is commonly used to determine the quantity of other proteins by comparing an 88 unknown quantity of protein to known amounts of bovine serum albumin (BSA). Due to BSA 89 having high stability, low cost, and a lack of effect in many biochemical reactions, it has 90 served many uses as a carrier protein, as a stabilizing agent in enzymatic reactions, and in 91 gel shift assays. These attributes serve as an excellent cross-selective template study for 92 Hb.

93
The aim of this paper is to optimise synthetic hydrogel-based MIPs to specifically recognise 94 and discriminate between species of proteins for future electrochemical diagnostic devices.

95
The application of protein-specific MIPs along with multivariate analysis offers the potential

269
Using hierarchical cluster analysis (HCA), a dendrogram was constructed to demonstrate the 270 interspecies homology using an optimised MIP system (Fig. 3). Considering the high 271 similarities between the proteins, specific MIPs are able to successfully discriminate between   (Fig. 5 a) and a non-imprint 309 control (NIP) (Fig. 5 b). It can be seen that the currents of the cycles decrease rapidly with 310 the number of cycles, which is attributed to the non-conducting (insulating) PAM membrane 311 layer formed on the electrode surface. This is especially true for the MIP (Fig. 5 a)  can be seen in Fig. 6  system is unable to discriminate between samples (Fig. 6 b), the BHb MIP system is 367 successfully able to discriminate/ between its native BHb template spiked within a mixture of 368 pig and human haemoglobins in biological samples (Fig. 6 a). These results suggest that 369 these MIP systems could be used for future biosensor development that relies on

475
PHb; 100 µg mL -1 each, are noted as U1, P1 and S1 respectively); Samples spiked with a 476 mixture in the absence of the original BHb template (HHb, PHb; 100 µg mL -1 each, are noted 477 as U2, P2 and S2 respectively). For demonstrative purposes, Both MIP and NIP plots were 478 kept at the same scale to illustrate the profiling pattern recognition effect.