Several coronaviruses from the family of Coronaviridae used to cause mild respiratory diseases until the severe acute respiratory syndrome coronavirus (SARS-CoV) was transmitted from animal to human and caused the severe acute respiratory syndrome (SARS) disease in 2002 [1]. In December 2019, a cluster of patients with severe respiratory syndrome (pneumonia) was reported in Wuhan, China, and the associated pathogen was identified as a novel coronavirus [2], which was later named as SARS-CoV-2, and the disease was named as COVID-19. Since then, the COVID-19 cases have been reported in every continent including Antarctica. On 11 March 2020, the World Health Organization (WHO) declared the COVID-19 as a pandemic [3]. As of 15 November 2021, the total number of COVID-19 cases reported by WHO is more than 252 million with an associated death of more than 5 million individuals [4]. This novel coronavirus is the seventh edition in the Coronaviridae family of viruses that causes respiratory diseases in humans. The spreading capability of the new virus (SARS-CoV-2) is higher than all the other coronaviruses and new variants of SARS-CoV-2 spread even faster in humans than the original virus. SARS-CoV-2 has glycosylated spike (S) proteins [5] that binds to the angiotensin-converting enzyme II (ACE2) receptor found in lower respiratory tracts of humans [6]. The virus can survive on plastic, metal, or cardboard surfaces for 4 h up to 3 days, making it easier for the virus to spread [7]. To minimize the rapid spread of SARS-CoV-2, effective viral detection methods are necessary. The most popular method of SARS-CoV-2 detection is the real-time reverse transcription-polymerase chain reaction (rRT-PCR). However, it has several limitations such as complex protocols, the need for trained personnel, long turnaround, and false-negative results [8]. Different detection techniques have been developed since 2019 including plasmonic-based colorimetric assays, antibody capture, and antigen-binding assays, paper-based biomolecular sensors, and thermoplasmonic chips [9]. However, there is still a need to develop effective, highly selective, and easy-to-use detection methods for SARS-CoV-2.

Recently, polymeric materials such as polydiacetylene (PDA) that have colorimetric responses to environmental stimuli have gained great interest in developing colorimetric biosensors [10,11,12,13,14]. Previous studies suggested that PDAs have demonstrated colorimetric transition responses to various bacteria and viruses [11, 15]. Charych et al. (1993) first reported a PDA-based sensor that could detect hemagglutinin of the influenza virus [16]. Lately, PDA-based sensors have been reported to detect influenza virus, hepatitis B, foot-and-mouth virus, bovine viral diarrhea virus, feline calicivirus, and vaccinia virus [15, 17, 18], suggesting a potential of using PDAs as a colorimetric sensor for coronavirus detection [19,20,21]. To the best of our knowledge, no report has been published that describes a PDA-based colorimetric sensor to detect SARS-CoV-2.

In this study, a PDA-based flexible biosensor was developed for detecting the S protein of SARS-CoV-2, resulting in biosensors for SARS-CoV-2. The anti-N is an antibody protein of nucleocapsid (N) structural protein of SARS-CoV-2 that is responsible for the viral replication in host cells [22]. The anti-N protein is more sensitive to detect early COVID-19 infection in comparison to the antibody protein specific to the spike protein [23]. Therefore, the PDA-based flexible biosensor was prepared by electrospinning PDA nanofiber composite with the conjugation of SARS-CoV-2 anti-N protein. The carboxylic acid group (–COOH) in the PDAs was transformed into an open ester bond (–COO–) that provided a binding site for the anti-N. When the anti-N conjugated PDA nanofiber (PU-PDA-NHS-anti) was introduced to the spike protein of the SARS-CoV-2, the nanofibers demonstrated a colorimetric transition from blue to red after incubation and the intensity of the color of red was increased after 24 h of incubation. In a comparison with no color changes found in the non-functionalized PU-PDA fibers, the functionalized PU-PDA-NHS-anti fibers showed colorimetric responses to the S protein of SARS-CoV-2. The anti-N proteins in the PU-PDA-NHS-anti fibers probably interacted with the S protein which triggered the color change in the fiber. Because the PDA nanofibers were flexible and lightweight and did not require a power supply such as a battery when the colorimetric detection to S protein occurred, the PDA nanofibers could be used in developing wearable devices for detecting SARS-CoV-2. Wearable devices, which allow physiological signals to be continuously monitored, can be used in the early detection of asymptomatic and pre-symptomatic cases of COVID-19 [24]. Wearable devices such as facemasks and medical gowns using the PDA nanofiber composites can be potentially developed to monitor in real time the contamination of SARS-CoV-2.

To prepare the PU-PDA-NHS-anti nanofiber biosensor, at first, the 10,12-pentacosadiynoic acid (PCDA) monomer was chemically functionalized to PCDA-NHS (Supplementary Document). During this functionalization, the carboxylic acid groups (–COOH) of PCDAs were transformed into open ester bonds (–COO–). To confirm the functionalization, a Nicolet 6700 Fourier transform infrared spectroscopy (FTIR) spectrometer (Thermo Electron Corporation, Madison, WI, USA) was used to collect attenuated total reflection-FTIR spectra of the PCDA and PCDA-NHS powders. The FTIR spectra of PCDA-NHS powder had a peak at wavenumber 1723 cm−1 that corresponds to ester bond (–COO–). This peak was not found in PCDA powders. The PCDA powders showed a peak at wavenumber 1689 cm−1 corresponding to carboxylic acid (–COOH) stretching typical in PCDA [15] (Fig. 1).

Fig. 1
figure 1

FTIR spectra of PCDA powders and PDA fiber samples

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Then, PCDA and PCDA-NHS powders and polyurethane (PU) matrix polymer were used to prepare PU-PDA-NHS fiber mats via electrospinning technique (Supplementary Document). Additionally, control samples of PU-PDA fiber mats were prepared by the procedures described in our previous report [11]. Both PU-PDA and PU-PDA-NHS fiber mats were blue (Fig. 2a). The fiber mats were peeled off from the aluminum foil as shown in Fig. 2b. Fibers were highly stretchable and curled up after peeling from the aluminum foil, suggesting good flexibility (Fig. 2c).

Fig. 2
figure 2

Images of a PDA nanofiber mat, b fiber mat during peeling off the aluminum foil, and c demonstration of high stretchability

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FTIR spectra of PU-PDA and PU-PDA-NHS fibers exhibited a similar trend as PCDA and PCDA-NHS powders. PU-PDA fibers showed a peak at wavenumber 1691 cm−1 (–COOH) that was not present in PU-PDA-NHS fibers. Instead, a new peak at wavenumber 1725 cm−1 (–COO–) was observed in PU-PDA-NHS fibers (Fig. 1). The results confirmed the esterification of PCDAs as a binding site for the anti-N protein [15, 18]. Finally, PU-PDA-NHS fiber mats were incubated with a solution containing 2 μg/mL anti-N protein for 48 h at 4 °C to prepare PU-PDA-NHS-anti fiber mats (Supplementary Document). No significant difference was found between the PU-PDA-NHS and PU-PDA-NHS-anti fibers when comparing the FTIR spectra (Fig. 1).

To confirm the anti-N conjugation occurring to the PU-PDA-NHS fibers, FTIR spectra were collected for PDA-NHS powders without PU. PCDA-NHS powders were photopolymerized and then UV irradiated at 254 nm for 3 min, resulting in PDA-NHS powders. The PDA-NHS powders were incubated with anti-N and FTIR spectra were taken before and after the incubation. The result showed a significant increase in the N–H stretching peak (3360 cm−1) (Fig. 3 arrow marked), arising due to the presence of nitrogen (N) in anti-N protein composition. The presence of N–H stretching peak confirmed the successful anti-N conjugation with the PDA-NHS (Fig. 3).

Fig. 3
figure 3

FTIR spectra of PDA-NHS and PDA-NHS-anti powders

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After the preparation of PU-PDA-NHS-anti nanofiber biosensor, circular fiber samples were cut to fit inside a 48-well plate. The fiber samples were washed using sterile water and PBS before they were incubated in SARS-CoV-2 S protein (SinoBiological, Wayne, PA) solution (1 μg/mL) using a low attachment 48-well plate at room temperature. The S protein preparation method with the buffer solution concentration can be found in the Supplementary Document. The S protein solution was diluted using a dilution factor suggested by the manufacturer. All the results reported here were obtained using the dilution factor of 1:500 that determined the protein concentration was 1 μg/mL. Previous report from our lab confirmed that PDA nanofibers do not change color at temperature less than 60 °C [10], so temperature during testing condition did not affect the colorimetric or morphological properties of the PDA nanofibers. Control samples of PU-PDA and PU-PDA-NHS fibers were also incubated with S protein solution. Morphological analysis of the fibers was conducted before and after the S protein incubation using scanning electron microscopic (SEM) images. A JEOL JSM-6500 field emission scanning electron microscope was used to collect SEM images. The results showed that PU-PDA fibers had a scale-like structure on the fiber surface which confirmed the attachment of PDAs on the PU fibers [11, 12] (Fig. 4). PU-PDA-NHS fibers had a similar morphology on the surface (Fig. 4). The PU-PDA-NHS-anti fibers had a similar structure but had some dot-like structures on the surface, which might be the anti-N proteins because this fiber was incubated for 48 h in the anti-N protein solution (Fig. 4, inset). The size of these dot-like structures varied from 50 to 700 nm, indicating different degrees of coagulation of antibody protein on the fiber surface. After 24 h of incubation with S protein, the PU-PDA fiber morphology remained the same, suggesting no chemical reaction between the fiber and S protein (Fig. 4). In comparison, the PU-PDA-NHS and PU-PDA-NHS-anti fibers showed a very rough surface after incubation with S protein (Fig. 4). Larger scale-like structures were observed in the fibers. The results indicated the topographical changes in the fiber morphology might be due to the interaction between the fibers (PU-PDA-NHS and PU-PDA-NHS-anti) and S protein.

Fig. 4
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SEM images PDA fibers before and after incubation with S protein

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From the SEM images, fiber diameters were measured using the ImageJ software. Ten fiber samples from each category were analyzed for fiber diameter analysis before and after incubation with S protein. The average diameter of PU-PDA nanofibers was 1700 nm. The PU-PDA-NHS and PU-PDA-NHS-anti nanofibers had average diameters of 1200 and 1300 nm respectively. The reduction of diameter in PU-PDA-NHS and PU-PDA-NHS-anti fibers was likely due to the addition of functionalized PCDA-NHS in the electrospinning solution. While electrospinning, the fibers were deposited randomly on the collector plate due to the high electrostatic force. So, the size of the nanofibers was not same for all the fibers, but rather had a range of diameters as shown in Fig. 5. The variation in diameter was not drastic compared to the previously reported PDA nanofibers prepared via electrospinning [11, 13, 25]. To evaluate statistically significant differences in the fiber diameters, analysis of variance (ANOVA) and Tukey’s honest significant difference (HSD) tests were performed. The results (Fig. 5) indicated that there was no significant difference between PU-PDA fibers before and after S protein incubation, which is in good agreement with the morphological observation of the fibers and indicated that the S protein did not react with the PU-PDA fibers. The diameters of the PU-PDA-NHS and PU-PDA-NHS-anti fibers were reduced after S protein incubation, which was likely due to the interaction between PDA fibers and S proteins. However, the diameter reduction in PU-PDA-NHS fiber after S protein incubation was not statistically significant. Fiber morphology and fiber diameter may change due to changes in electrospinning parameters [26], and addition of surfactants, chemicals, and drug molecules [27], as well as external stimuli [28]. Previous studies reported a diameter increase and a scale-like morphological change in the PU-PDA fibers in comparison with pristine PU fibers [11]. When the PDAs were modified to prepare PU-PDA-NHS and PU-PDA-NHS-anti fibers, the scale-like structure was also observed (Fig. 4). Then, after the fibers were incubated with S protein, the scales on fiber surface were disrupted as shown in Fig. 4. A hypothesis was that the changes in fiber surface morphology were due to the interaction between antibody conjugated PDA-NHS and S protein. Figure 4 shows no changes found in the PU-PDA fiber after incubation with S protein. It was likely because there was no interaction with the S protein due to the absence of PDA-NHS or PDA-NHS-anti. The nature and dynamics of the interaction between PDA-NHS-anti molecules in the fibers with S protein of SARS-CoV-2 needs further experimental analysis.

Fig. 5
figure 5

PDA fiber diameter before and after incubation with S protein. ANOVA and HSD were performed for statistical analysis (*, ***, and **** indicate p ≤ 0.05, p ≤ 0.001, and p ≤ 0.0001)

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When the PU-PDA-NHS-anti fiber was incubated with S protein (1 μg/mL) at room temperature, the fiber started changing color from blue to red after 4 h of incubation. The color of red became bright after 24 h of incubation (Fig. 6a). In addition, PU-PDA and PU-PDA-NHS fibers were incubated with the S protein. No color change was observed in PU-PDA fibers as expected, which was in good agreement with the morphological observation and fiber diameter analysis. Furthermore, a significant color change was observed in PU-PDA-NHS fiber after 24 h of incubation with S protein (Fig. 6a). That was because the PU-PDA-NHS fiber had open ester groups (–COO–) on macromolecules, and hence some S proteins might have been able to conjugate to the PU-PDA-NHS fiber, resulting in a color change. Additionally, all the fibers were incubated with the buffer solution that was used to prepare the S protein solution and no color change was observed. The buffer solution was composed of 0.1% bovine serum albumin (BSA) protein in wash buffer (Supplementary Document). The PDA fibers did not change color when they were incubated with buffer solution, thus signifying a specific detection of S protein.

Fig. 6
figure 6

a Photographs of PDA fibers before and after 24 h of incubation with S protein, b ΔE values of PDA fibers

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The color change in the fibers was quantified using CIE (International Commission of Illumination) color space coordinates that include the values of L*, a*, and b* representing lightness, color position between green and red, and color position between yellow and blue respectively [29]. Photographs of PDA fibers were taken before and after 24 h of incubation with S protein (Fig. 6). CIE L*a*b* values were obtained from the photograph using Digital Color Meter application (Apple Inc.). Twenty values of L*, a*, and b* were collected for each fiber on the photographs. Ten values were for samples before incubation and 10 values were for after incubation. The color change of the fibers was quantified by calculating the ΔE values using Eq. (1).

$$\mathrm{{\Delta}E}=\sqrt{{(\mathrm{{\Delta}L})}^2+{(\mathrm\Delta\mathrm a)}^2+{(\mathrm{{\Delta}b})}^2}$$
(1)

where ΔL, Δa, and Δb are the differences in brightness, redness, and yellowness of the fibers before and after the incubation with S protein [29], respectively. The ΔE values are shown in Fig. 6b. The ΔE for PU-PDA was 17.61, which is very low compared to the ΔE of PU-PDA-NHS (70.52) and PU-PDA-NHS-anti (78.49) fibers. The higher ΔE of PU-PDA-NHS and PU-PDA-NHS-anti fibers confirmed a distinguishable color change in the fibers after incubation with S protein. Additionally, it was observed that the difference in the ΔE between PU-PDA-NHS and PU-PDA-NHS-anti was very minimum, suggesting that the antibody functionalization may not be necessary for S protein detection. The removal of antibody conjugation can offer cost reduction in biosensor preparation; however, because the color change in PU-PDA-NHS fiber was not proven specifically due to the open ester bond, more control experiments need to be conducted in the future to fully understand the specificity of the fiber.

The average L*a*b* of the fibers before and after incubation with S protein were measured. A negative a* value represents green region but positive a* represents red, and a negative b* indicates blue whereas a positive b* indicates yellowness [30]. b* of all fibers were negative before S protein incubation, meaning the fibers were blue. However, after the incubation, only PU-PDA fiber’s b* remained negative (− 5.749) which indicated the fiber color remained blue after S protein incubation. On the other hand, a* values of PU-PDA-NHS (44.341) and PU-PDA-NHS-anti (48.614) became positive after S protein incubation, indicating the color of these fibers shifted to red.

The vivid blue to the red transformation of the PU-PDA-NHS-anti fiber suggested an interaction between the S protein and the anti-N protein that might have been responded by a perturbation in the electron cloud of the PDAs and resulted in a color change. The nature of this protein–protein interaction between anti-N and S proteins will be investigated in the future. The initial indication of this protein–protein interaction can help to prepare targeted therapeutics and efficient biosensors that can effectively sense and inactivate the SARS-CoV-2 in the respiratory cells. The preliminary results that were illustrated here have demonstrated that the antibody conjugated PDA nanofibers have the potential in creating wearable devices and personal protective equipment such as face masks and medical gowns for real-time detecting and monitoring SARS-CoV-2 contamination and contraction on a daily basis as well as in a healthcare setting. Further investigation will focus on sensitivity, selectivity, and biosafety of PDA’s colorimetric responses to S protein, which are critical in proof-of-concept of using PDAs in SARS-CoV-2 detection techniques. Additional studies such as comparability and durability of the flexible PDA nanofibers should be considered with the textile materials commonly used in personal protective equipment such as facemasks and medical gowns. A viral load model was described by Soto et al. (2021), where a 4-h timeframe provided a sufficient threshold number of virus captured by a functionalized face mask to perform analytical analysis [31]. The PDA nanofiber biosensor described in this work exhibited a color change after 4 h of incubation with the S protein of SARS-CoV-2, which was consistent with the timeframe reported by Soto et al. (2021) [31]. The 4-h timeframe is a good indication of mask wearing base requirement for an effective sensing of the virus via a mask protection. Future work would require a detection study with live SARS-CoV-2 as well as an optimization study of electrospinning parameters and antibody concentration in the fiber to improve the sensitivity of the PDA nanofibers.

PDA nanofibers were obtained via chemical functionalization of PCDA monomer and electrospinning, resulting in a binding site for a receptor molecule—nucleocapsid antibody (anti-N) protein of SARS-CoV-2. The anti-N protein was attached to the functionalized PDA fibers via conjugation, resulting in PU-PDA-NHS-anti fibers. The PU-PDA-NHS-anti fibers showed a colorimetric transition from blue-to-red when interacting with S protein of SARS-CoV-2 at room temperature, suggesting it was a colorimetric detection of SARS-CoV-2. The results demonstrated the potential of using PDA nanofibers in creating a flexible, lightweight, no-battery, and colorimetric biosensor. The PDA biosensors can be used in developing wearable devices and personal protective equipment such as face masks and medical gowns, providing real-time detection to COVID-19 contraction and hence timely medical decisions to improve public health in pandemics. In addition, the flexible PDA biosensors can be used in hygiene products, food processing, and food packaging, providing real-time detection via colorimetric indication. These smart products will empower individuals in the decision-making process and improve health and well-being.