Colorimetric examination is a common analytical method for measuring chemicals in both research and clinical fields. The principle of this method is to observe the colour changes of a mixture solution of the specific analyte and the recognising agent due to their chemical or biochemical reactions [
1,
2,
3,
4,
5]. The operator can read the quantitative result of the chemical presence by observing the colour changes of the mixture with the naked eye [
1,
3,
4,
6,
7]. Thanks to the rapid results, friendly operation, and simple storage, the colorimetric detection method has been used widely in society, such as the haemoglobin blood test, glucose test strip, and urine test strip, or commercial test kits for solution-based tests. Of these tools, commercial test kits are being used for research and in clinical laboratories due to their high sensitivity, various analyte tests, and especially, quantitative results. The effective accessory supporting commercial test kits is the microplate, which can be described as a pattern of microwells, equipped on a flat platform. The microplate supports the measurements of multiple samples at the same time, saving time and effort for the users. There are several types of microplates with various numbers of wells, i.e., 6, 24, 48, 96, or 384 wells, and different colours, i.e., black, white, or clear [
8,
9]. Currently, the 96-well microplate (96-WM) is the plate most used in research and clinical facilities as it provides more options for the number of tests and has standard dimensions for use with commercial testing instruments [
10,
11]. Although they can support multiple tests simultaneously, 96-WM reading requires specific equipment to examine multiple wells in the plate, which is usually cumbersome and non-portable, and this causes the operators to remain in a laboratory environment [
12,
13,
14]. In recent years, researchers have focused on develo** portable devices, which can examine the microplate reading to support the measurement in the remote testing conditions. In 2016, Feng et al. developed a portable 3D-printed device using a smartphone to perform the antimicrobial susceptibility testing (AST) on a 96-well microplate, targeting the gram-negative bacteria,
Klebsiella pneumoniae with the measurement accuracy being over 95% [
11]. In the same year, Fu et al. introduced another smartphone-based device, using commercial chemicals to measure various biomarkers spiked in blood and urine samples [
12]. Their device showed good results in the limit of detection of 17.54 U/L for alanine aminotransferase, 15.56 U/L for alkaline phosphatase, 0.00135 mmol/L (1.35 μM) for creatinine spiked in urine samples, and other biomarkers. Other researchers also reported smartphone-related devices developed using microplate with clinical-approved testing kits to detect particular biomarkers of infectious diseases, such as varicella zoster virus IgG (VZV) for detecting chicken pox (varicella) and shingles (herpes zoster), cytomegalovirus IgG (CMV) for detecting herpes viruses, or the protein CFP-10 for detecting tuberculosis [
13,
15], female reproductive steroid hormone profiles [
14], or the progesterone concentration in the whole blood sample [
16]. Although these devices reported high sensitivity and stability, most are only used in research applications. In these devices, a smartphone is commonly used as the optical sensor, but the rapid development of mobile technology and the varied dimensions of smartphones create compatibility issues, and the internal processing of the optical cameras affect measurement accuracy [
5]. Furthermore, smartphone use introduces issues with user confidentiality and cross-contamination hazards [
5,
17].
Another parameter for consideration when develo** a chemical analysis device is the target analyte. As mentioned above, portable colorimetry devices can detect various chemical or biomolecules in solutions, which can be used as biomarkers, such as the presence of albumin in urine indicating kidney disease, a reduced citrate level in urine suggesting prostate cancer, the level of cardiac Troponin I (cTnI) in blood hel** the diagnosis of acute myocardial infarction, or the absence of glutathione in saliva indicating the potential of head-neck cancer [
5,
18,
19,
20,
21]. The detection target will influence the design choices, such as the sensitivity of the detecting sensor, the background light intensity, and the recognising reagent type. In this research, urine has been chosen as the target biofluid due to its clinical importance in healthcare and the non-invasive method of collection. The chosen biomarkers are creatinine and glucose, which are two of the common biomarkers tested in human urine examination. In normal conditions, creatinine in urine varies from 2.5 to 17 mmol/L (28.28 to 192.304 mg/dL), while glucose is not present in urine [
22]. The presence of glucose in urine is a sign of biological dysfunction, whilst a high concentration of creatinine may be an indication of diabetes, muscle damage, or kidney disorder [
23,
24]. In clinical urine tests, creatinine and glucose in urine samples can be first observed by using dip sticks. However, the dip-stick method only provides initial screening; therefore, medical staff often measure them on automated analysers using colorimetric or enzymatic assays with high accuracy and reliability. If the measurements from these methods can be equipped into a compact portable device, it will provide potential for use in rural and remote areas.
In this report, we have developed a portable colorimetry device to facilitate a 96-WM with a commercial test reagent. The targets of detection biomarkers in this research are glucose and creatinine, two common biomarkers present in human urine. The device was tested with two commercial test reagents, and its performance compared firstly with results from a clinical microplate reader, and secondly, using urine samples from renal patients and comparing the results with clinical results provided by a local pathology laboratory.