Biosensors play an important role in clinical diagnostics, point-of-care testing, personalized medicine, and pharmaceutical research [
1]. In this area, highly sensitive detection systems which have excellent specificity are required for their ability to provide useful insights into an individual's health [
2]. Over the last decade, the scientific community has focused much of its research on biosensor systems applicable to point-of-care (POC) diagnostic devices which can rapidly assess the cause of various illnesses among patients [
1,
2,
3,
4,
5]. These diagnostic devices facilitate faster and accurate identification of diseases, which lead to better treatment of patients [
1,
2]. Among these devices, hydrogels are attracting attention due to their versatility to chemical modification which makes them responsive to external stimuli such as pH [
6,
7,
8,
9], or temperature [
1,
10] and the prospect of encapsulating therapeutic agents such as antimicrobials like zinc and silver containing nanoparticles within their matrix [
2]. They are developed from both synthetic polymers and biopolymers, and are utilized in tissue engineering, artificial biomedical scaffolds, soft actuators, wound dressings and environmental remediation [
6,
11]. Chitosan [
12,
13], cellulose [
14,
15,
16], pectin [
17], alginate [
18,
19], and carrageenan [
20,
21] are the most attractive surrogates to petroleum based starting materials due to their relative abundance and availability. Moreover, their biodegradability, biocompatibility, ease of functionalization and gelling properties makes them ideal starting materials for developing hydrogels [
22,
23]. These features can also be enhanced through chemical and physical crosslinking of two different hydrogels to create dual crosslinked polymers [
24,
25]. Incorporation of conjugated polymers and other chemical moieties within their matrices further enhances their functionality as they impart the ability to respond to an external stimuli [
2,
26,
27]. Conjugated polymers such as polyaniline, polypyrrole, and polydiacetylene (PDA) enable them to function as electrochemical biosensors since the electrochemical properties of the conjugate systems are associated with visible colorimetric changes [
26,
28]. PDAs are especially attractive as they exhibit a blue to red colour transition visible to the naked eye when they are subjected to external stimuli such as changes in temperature, pH, bacterial cells, and aromatic compounds [
7,
29]. They have also been used in the development of highly sensitive colorimetric probes for the detection of cholesterol [
5], ammonia [
30], glucose [
31], microorganisms [
7,
32], volatile organic compounds [
33], active pharmaceutical excipients, small and large biomolecules among other compounds [
28]. In this study, a hydrogel prepared from polydiacetylene-zinc oxide-carboxymethyl chitosan-hydroxyethyl cellulose (PDA-ZnO-CMCs-HEC) was evaluated for its pH responsiveness, colorimetric transitions and inhibitory activity against
E. coli. First, chitosan (Cs) was esterified with monochloroacetic acid to obtain carboxymethyl chitosan (CMCs) which was subsequently chemically crosslinked with hydroxyethyl cellulose (HEC) using citric acid as the crosslinking agent to obtain CMCs-HEC hydrogels. To enhance the pH responsiveness of the hydrogels, PDA-ZnO was introduced into the CMCs-HEC hydrogel to obtain a colorimetric pH responsive PDA-ZnO-CMCs-HEC hydrogel. To better understand the properties such as pH responsiveness, thermal profile, crystallinity, functional group changes upon crosslinking and antimicrobial activity of the hydrogels against
E. coli, the hydrogels were then analysed using a Fourier transform infrared spectrophotometer (FT-IR), powder X-ray diffraction (XRD), scanning electron microscopy (SEM), thermal gravimeter analyser (TGA) and UV-Vis techniques as well as by antibacterial assays.