A century ago, the Nobel Prize in Physiology or Medicine was awarded jointly to Frederick Grant Banting and John James Rickard Macleod for the discovery of insulin and its relationship with diabetes [1]. Even though this discovery has saved millions of lives, according to the International Diabetes Federation (IDF), one person dies of diabetes every 5 s worldwide [2]. IDF statistics for 2021 show that 537 million adults (20–79 years) are living with diabetes, nearly 1 in 10, and this number is predicted to rise to 643 million by 2030 and 783 million by 2045 [3]. Diabetes mellitus is a chronic metabolic disorder characterised by the inability of the β-cells of the pancreas to produce enough insulin [4]. In Type I diabetes, the pancreas is incapable of producing insulin, while in Type II diabetes, the pancreas does not produce enough insulin and/or the body becomes resistant to it. Type II diabetes is treated with insulin while Type II diabetes can be treated with drugs that lower glucose independently of insulin. Eventually, many Type II diabetic patients also become reliant on exogenous insulin for regulating their glucose levels.

Diabetes management requires the regular checking of blood glucose levels and, for more than four decades, glucose biosensors have been in use by diabetic patients ^[5][6][7][8][5,6,7,8]. However, measuring glucose is an indirect method for calculating the required insulin dosage and failure to administer the correct amount of insulin can lead to severe hypoglycaemia or hyperglycaemia with serious consequences to health. Continuous insulin and glucose monitoring would enable a more accurate estimation of insulin sensitivity, regulate insulin dosage and facilitate progress towards the development of a reliable artificial pancreas ^{[9][10][11][12][13]}[9,10,11,12,13]. Although several insulin detection devices have been developed, many challenges remain, associated with establishing specificity, enabling detection at the nanomolar range and avoiding interference from endogenous molecules. At present, the needs for miniaturised, low-cost, easy-to-use and reliable insulin biosensor platforms remain largely unmet. Several methods suitable for point-of-care insulin detection, mainly direct electrochemical and optical methods such as fluorescence, are reviewed. The challenges and limitations regarding specificity, sensitivity and appropriate detection limits of human insulin are explored. Nucleic acids such as aptamers, molecularly imprinted polymers and nanomaterials like modified carbon nanotubes [14] that have high affinity and specificity towards insulin offer promising exciting possibilities for enhancing sensitivity, lowering detection limits and improving insulin biosensor stability.

2. Insulin Structure and Function and Its Detection in Biological Fluids

Insulin is a 5808 Da peptide hormone produced and secreted by the pancreas in response to increased levels of glucose in the circulation. It consists of a 21-amino-acid A chain and a 30-amino-acid B chain held together by two disulfide bonds and is responsible for regulating carbohydrate, lipid and protein metabolism by stimulating the uptake of glucose through insulin receptors found mainly in peripheral muscle, in adipocytes and in hepatocytes. Impaired insulin production and secretion and/or a reduced response to insulin, also known as insulin resistance, are key underlying mechanisms leading to the development of type II diabetes ^[15][16][15,16]. To compensate for the resistance to insulin, the pancreas secretes more insulin into the circulation (hyperinsulinaemia). Although moderate levels of insulin resistance can be beneficial for ensuring the supply of glucose to the brain [17], exacerbated levels of resistance can result in chronic hyperglycaemia, leading to prediabetes and type II diabetes. Insulin resistance can be evident long before the development of diabetes and thus elevated fasting levels of insulin can potentially predict the onset of this metabolic disorder [18]. Insulin can be measured in blood serum and plasma, but it can also be detected in other biological fluids such as saliva, tears or sweat ^[19][20][21][19,20,21]. Measurement of insulin is, however, challenging mainly because it is present at very low concentrations in serum and plasma, and this requires very sensitive techniques (Table 1). At normal fasting conditions, the insulin concentration in serum can be below 50 pmol/L while concentrations of >70 pmol/L indicate insulin resistance and the onset of type II diabetes ^[22][23][22,23]. In interstitial fluid, insulin concentrations are on the order of 20–50% lower than those in plasma ^[24][25][26][24,25,26]. Similarly in saliva, insulin levels were shown to be consistently lower than those of plasma ^[20][27][28][20,27,28]. Fabre et al. [20] studied saliva in children where insulin levels were as low as 10% of those found in plasma. Messenger et al. [28] reported even lower salivary insulin levels than plasma close to 50%.