Biomolecule self-assembling is highly implicated in various biologic events through a variety of non-covalent interactions, such as hydrophobic interaction, hydrogen bonds, electrostatic interaction, and metal coordination (Bera et al., 2020; Yu et al., 2020). The self-assembly structures are the basis of forming different complex biological structures (Cao et al., 2020). For example, the cell membrane is mainly an elastic semi-permeable membrane composed of amphiphilic phospholipids (small organic molecules with a hydrophilic phosphate heard and a hydrophobic long alkyl chain tail) (Momcilovic et al., 2020). Inspired by the self-assembly events in living organisms, scientists have prepared many functional supramolecular systems through the self-assembly of organic small molecules in recent years (Ballandras-Colas et al., 2017; Chen et al., 2018; Vantomme and Meijer, 2019). Among them, the amphiphilic diacetylene-based self-assembly systems have attracted great attention in the field of basic and applied research (Lu et al., 2001; Peng et al., 2009; Hu et al., 2014; Chae et al., 2016; Ishijima et al., 2017a; Jordan et al., 2017; Nsubuga et al., 2018; Ortiz-Cervantes et al., 2018). Amphiphilic diacetylenes not only have the self-assembly properties similar to phospholipids, but more importantly, it can form a conjugated structure through polymerization (Wang et al., 2017). The covalently connected conjugated structures can give PDAs far beyond the stability of common supramolecular self-assembly (Park et al., 2016a; Tian et al., 2020). The stable structure is very important for biological detection and bioimaging, especially for complex environments in organisms.

A series of amphiphilic diacetylene molecules with different polar heads and tail groups can be prepared by the Cadiot-Chodkiewicz reaction using acetylene or halocetylene derivatives (Reppy and Pindzola, 2007). The topochemical polymerization takes place among the arranged diacetylene amphiphiles under excitation of the ultraviolet light or gamma ray, followed by the formation of polymer conjugated skeletons with alternating C=C and C=C bonds (Figure 1) (Reppy and Pindzola, 2007; Li et al., 2018). The special conjugated main chain of PDA gives it unique C=C Raman signal, strong absorption at 640 nm, and fluorescence properties. PDAs are easy to be functionalized and can specifically bind to specific biomolecules. The absorption spectra of the functionalized PDAs would migrate from 640 to 540 nm after binding. This process will cause the color change of PDA from blue to red, coupled with the appearance of red fluorescence. These characteristics make PDA very attractive for applications in biological detection and bioimaging.

In 1993, Charych et al. reported the first case of modified PDAs for detection of virus (Charych et al., 1993). During the following 20 years, many PDA-based bioprobes have been developed to detect biomarkers, biomolecules, metal ions, drugs, proteins, nucleic acids, lipids, virus, bacterial, and use to bioimaging (Chae et al., 2016; Park et al., 2016b; Qu et al., 2016; Yamamoto et al., 2016; Jiang et al., 2017; Cui et al., 2018a; Kang et al., 2018; Nsubuga et al., 2018; Stauber et al., 2018; Takeuchi et al., 2018; Heo et al., 2019; Jung and Park, 2019; Kim and Lee, 2019; Oh et al., 2019; Romero-Ben et al., 2019; Wang et al., 2019a; Zhang et al., 2019a; Adhikary et al., 2020; Fang et al., 2020; Hao and Zhu, 2020; Li et al., 2020; Tian et al., 2020). Our research group has also carried out some related work in this field. For example, we used imidazole-functionalized PDA nanosheets to detect lysophosphatidic acid (an early-stage ovarian cancer biomarker) in serum (Wang et al., 2017). In recent years, the preparation methods for PDA have been greatly advanced. PDA structures have been enriched from traditional films, micelles and vesicles to 3D-PDA structures, porous structures, hydrogels, macrocyclic structures, and single-layer nanosheets (Bang et al., 2016; Chae et al., 2016; Krishnan et al., 2016; Okaniwa et al., 2016; Jordan et al., 2017; Li et al., 2017a; Romera et al., 2017; Jeong et al., 2018; Takeuchi et al., 2018; Terada et al., 2018; Heo et al., 2019; Hu et al., 2019; Seo et al., 2019; Shin et al., 2020). Great progresses have also been made in the preparation of reversible and chiral PDA (Chen et al., 2016; He et al., 2020). PDA bioprobes based on these novel structures can further increase the sensitivity and reproducibility of detection, and their detection applications have thus been expanded to a wide range of biomolecules. For example, PDA colorimetric and fluorescent probes have been created to detect transmembrane action in the blood brain barrier (Adhikary et al., 2020), drug carrier efficiency (Wang et al., 2019a), chiral recognition (He et al., 2018) etc. In addition, PDA probes have also been extended to the fields of Raman bioimaging and Raman detection (Cui et al., 2018a; Tian et al., 2020). In this paper, we first introduce conventional and advanced preparation technologies of PDA. On this basis, the applications, and the latest progress of PDA in biological detection and bioimaging is highlighted. Finally, the challenges and opportunities for practical applications of PDA in biomedical field are discussed.

Generally, PDA bioprobes are prepared by functionalizing the PDA side chains with groups that can specifically bind to biological molecules. When biomolecules interact with PDA probes, the color of PDA probes will change from blue to red. In this way, biomolecules can be detected by colorimetry. It is therefore important to study the color change of PDA probes for the design of sensitive PDA probes. Alkyne carbons in diacetylene amphiphile monomers are sp hybrid with a bond angle of 180 degrees. Polymerization of diacetylene amphiphiles leads to the transformation of alkyne carbons from sp hybrid to sp². Under normal condition, the bond angle of sp² hybrid is 120 degrees. However, the force between the closely packed side chains makes PDA unable to rotate and change the angle of the PDA main chain during polymerization, resulting in stress in PDA. The biological interaction on the side chain of PDA sensors can cause the change of packed side chain, thus releasing the stress and enabling the main chain of PDA to rotate and change the color of PDA. Although the blue-to-red transition mechanisms of PDA are not fully elucidated, it is widely accepted that the absorption characteristics of the main chain are very sensitive to the change of the strain. Theoretical calculation showed that only several degrees of rotation in the PDA main chain would significantly change the π-orbital overlap and affect the electronic state (Hao and Zhu, 2020). The color change of PDA is fast and highly sensitive. As a result, the current biological probes based on PDA are widely used in biological detection (Reppy and Pindzola, 2007; Yoon et al., 2009; Sun et al., 2010; Chen et al., 2012; Lee et al., 2016b; Qian and Stadler, 2019; Zhang et al., 2019a; Hao and Zhu, 2020). We here introduce the applications of PDA in colorimetric bio-detection according to the size and type of detection targets, including small biomolecules, biomacromolecules, microorganism, drug screening, etc.

PDA are mainly composed of C-C-C, C=C, and C=C bonds, all of which have specific Raman scattering peaks. The intensity and position of Raman spectra of PDA derived from these specific bonds are closely related to the change of conjugated path length on the main chain of PDA. In the blue phase of PDA, the π-electron delocalization length of the main chain of PDA is large due to the restriction of the spatial arrangement of the side chain of PDA. The characteristic Raman peaks of C=C and C=C in PDA are located at 1,453 and 2,083 cm⁻¹, respectively. When PDA changes from blue phase to red phase, the arrangement restriction of the side chain of PDA is released, resulting in a shorter π-electron delocalization length of the main chain of PDA and greater vibration amplitude of C=C and C=C. As a result, the C=C and C=C bond vibrations absorb more energy, and the Raman peaks of C=C and C=C move to the larger wave number. In the red phase of PDA, the Raman peaks of C=C and C=C shift to 1,515 and 2,123 cm⁻¹, respectively (Wang et al., 2017).

Raman detection derived from PDA probes is very attractive in biological detection and imaging. Raman spectrum can provide rapid, simple, repeatable, non-destructive qualitative, and quantitative analysis. It does not need complicated sample pretreatment, and the samples can be measured directly through the optical fiber probe or through the transparent medium. Raman spectroscopy is particularly suitable for the detection or imaging of biological samples, because the Raman scattering of water is very weak, which can greatly eliminate the interference of the environment background. In addition, Raman detection has high selectivity, since molecules have their own Raman fingerprint peaks (Gan et al., 2019). However, such applications of PDAs are still in the infancy, presumably due to the weak Raman signal caused by the small Raman cross sections area of C=C and C=C in PDA (Jun et al., 2011; Kim H.-M. et al., 2015).

Recently, surface enhanced Raman scattering (SERS) and resonance Raman spectroscopy have been used to enhance the intensity of characteristic Raman signal of PDA. SERS technology can overcome the inherent signal limitation of traditional Raman spectrum. Its enhancement factor can be as high as 10¹⁴-10¹⁵ times, which is enough to detect the Raman signal of a single molecule. In 2018, Cui et al. assembled and polymerized PDA shell on the surface of silver nanoparticles (Cui et al., 2018b). They found that the characteristic Raman peaks of C-C-C, C=C, and C=C in PDA polymer were 83, 92, and 78 times higher than those in the thick PDA shell (about 20 ± 10 nm).

Compared with the SERS enhancement method, Resonance Raman Spectroscopy does not need to build the Surface Enhanced Raman Scattering base. Therefore, an drastic enhancement in the Raman intensity of PDA was also achieved by the Resonance Raman Spectroscopy technology to enhance the (Figure 10) (Cui et al., 2018a). When the wavelength of the incident laser matched well with the absorption wavelength of the conjugated polymer, Resonance Raman signals would be generated. The maximum enhancement ratio of the Raman signal could reach to 10⁷ times of the original. Specially, the maximum enhanced Resonance Raman can be obtained when the blue phase PDA is excited by 633 nm laser (the maximum absorption peak of the blue phase PDA). When PDA changed from blue phase to red phase, the excitation wavelength of PDA must be changed from 633 to 514 nm in order to obtain high Resonance Raman Spectroscopy enhancement effect. Raman detection of cyclodextrin was achieved by introducing ethylenediamine groups on the surface of PDAs. When cyclodextrin was present, the hydrogen bonding between the cyclodextrin and the side chain of PDAs caused the absorption of PDAs at 512 nm to increase greatly, and the Raman signal of PDAs could be greatly increased by 512 nm excitation. The PDA Raman probes based on the Resonance Raman Spectroscopy enhancement had very high sensitivity and could detect α-cyclodextrin at a low concentration of 0.01 mM.

It is worth noting that compared with other bioimaging methods, Raman imaging has the advantages of providing structure information of the detected biomolecules, narrow bandwidth, multi-color, multi-channel imaging analysis, among others. Proteins, nucleotides, lipids, sugars, and other biomolecules in vivo have strong background Raman signals in the range of 400–1,800 cm⁻¹ and 2,800–3,100 cm⁻¹. However, the Raman spectra of C-D, C=N and C=C bonds are located at 1,800–2,800 cm⁻¹, outside Raman window of these biomolecules. Therefore, many molecules containing C-D, C=N and C=C such as ethynylbenzene, 5-ethynyl-2′-deoxyuridine, diphenyl phosphorazidate, diphenylbutadiyne, benzonitrile, and 5-bromopentaninitrile, have been developed for Raman imaging to minimize the background Raman signals in vivo (Yamakoshi et al., 2011; Song et al., 2014; Li et al., 2017c). However, these small-molecule Raman probes often have very weak Raman signals and thus not sensitive enough for in vivo Raman bioimaging. The chain of PDA contains C=C. More importantly, the super long π conjugation of PDA can increase the Raman resonance of C=C. Therefore, PDAs are considered promising candidates for creating effective Raman imaging probes.

Very recently, Tian et al. developed a PDA probe for Raman imaging of living cells (Figure 11) (Tian et al., 2020). the PDA Raman probe was prepared by self-assembly of deca-4,5-diynedioic acid and bis (pyridyl) oxalamide-functionalized PDA were self-assembled into an ordered structure via the host-guest interaction, followed by UV polymerization. They found that the Raman intensity of the C=C bond of PDA was different upon excitation at 488, 532, and 785 nm. Under 785 nm excitation, the average Raman intensity of each C=C was 100 times that of 5-ethylyl-2′-deoxyuridine, benefiting from the conjugate structure of the main chain of PDA. The C=C Raman signal of PDA could further be enhanced through the Resonance Raman mechanism when the excitation wavelength was closed to the absorption peak of PDA. The average each C=C Raman intensity of PDA through the Resonance Raman mechanism was even 10⁴ times higher than that of 5-ethylyl-2′-deoxyuridine under 488 and 532 nm laser excitation. Thanks to the special conjugate structure of PDA and the Resonance Raman mechanism, high quality Raman microscopic imaging of living cells could be realized by PDA. The ultra-strong alkyne Raman signal of PDA could be used as reporters for living cells Raman imaging. Through the specific functional modification of PDAs (P2-P4), the C=C Raman signal (2,120 cm⁻¹) of PDAs can be used as a good Raman tag to image the lysosome (P2), mitochondrial (P3), and nucleus (P4) of living Hela Cells.

Aside from the unique colorimetric property, the fluorescence of PDA has also attracted scientists' interest for a long time (Kobayashi et al., 1997; Lécuiller et al., 1999; Yamamoto et al., 2016; Roh et al., 2017; Cui et al., 2018b; Wang et al., 2018b, 2020; Kim and Lee, 2019; Kim et al., 2019). Similarly, fluorescence of PDA will change when PDA transformed from blue phase to red phase. Kobayashi et al. reported that the fluorescence lifetime of blue-phase PDA was only 0.92 and 1.08 ps at 290 and 10 K, respectively (Kobayashi et al., 1997). The ultrafast relaxation of the excited state of blue-phase PDA resulted in its ultra-weak fluorescence intensity. The quantum yield of the blue-phase PDA was lower than 1 × 10⁻⁵. In comparison, the fluorescence lifetime of red-phase PDA was 52 ps at 15 K, much longer than that of blue-phase PDA (Lécuiller et al., 1999). The long relaxation time of the excited state could greatly increase the fluorescence of red-phase PDA, with a quantum yield of about 0.3. However, the fluorescence intensity of red-phase PDA at room temperature was much weaker than that at 15K, and the quantum yield of red phase PDA was about 0.02. Of note, the quantum yield of red-phase PDA was still much higher than that of blue-phase PDA at room temperature. Therefore, PDA could be considered as a good “turn-on” fluorescent probe.

The PDA-based fluorescent probes had many merits, such as high sensitivity, good selectivity, wide linear range, and little influence by external conditions. Therefore, PDA fluorescent probes have been widely used in biomolecular detection and cell imaging analysis in recent years (Wu et al., 2014; Zhang et al., 2014, 2018; Jiang et al., 2017). For example, Roh et al. developed a PDA-based fluorescent probe for hepatis B surface antigen (Figure 12) (Roh et al., 2017). Moreover, a convenient and economical fluorescent probe was prepared by loading the Hepatitis B surface antibody-modified PDA onto nitrocellulose test paper. In the presence of hepatitis B surface antigen, PDA would undergo blue-to-red phase transformation and emitted red fluorescence. In this way, the detection of hepatitis B surface antigen could be realized by both fluorescence and colorimetric detection with high sensitivity. However, the lowest detection limit of hepatis B surface antigen by the fluorescence method was 0.1 ng/ml, 10 times lower than that of colorimetry (1 ng/ml) under the same conditions, suggesting the higher sensitivity of PDA fluorescent probes.

To some extent, the low quantum yield of PDA fluorescence at room temperature limits applications of PDA fluorescence probes for biological detection and bioimaging. To address this limitation, Cui et al. developed an effective way to enhance the fluorescence of PDA by combining metal nanoparticles with surface plasmon enhancement effect (Cui et al., 2018b). They prepared Ag@PDA nanoparticles with a core-shell structure. The interaction between silver nanoparticles and PDA not only made the maximum fluorescence emission peak of PDA red shifted by 10 nm, but also made the fluorescence intensity of Ag@PDA nanoparticles enhanced by 7 times compared to pure PDA.

Apart from metal, the introduction of fluorescence energy resonance transfer mechanism into the detection can improve the fluorescence quantum yield of PDA. For example, Wang et al. prepared a fluorescent sensor containing PDA liposome composite for sialic acid detection by assembling and polymerizing three kinds of amphiphilic diacetylenes with the head group of phenylboric acid, 1,8-naphthalimide derivatives and ethylenediamine (Figure 13) (Wang et al., 2018b). In this PDA fluorescence sensor, the phenylboric acid head group could specifically bind with sialic acid and 1,8-naphthalimide derivative (the quantum yields >0.4) were fluorescence indicators. In the blue phase of PDA liposomes, the energy transfer between the fluorescence group of 1,8-naphthalimide derivative and the conjugated skeleton of PDA could quench the fluorescence of 1,8-naphthalimide derivative. In the presence of sialic acid, nevertheless, the blue-phase PDA would transform into the red-phase PDA, resulting in the fluorescence recovery of 1,8-naphthalimide derivative. The PDA fluorescence sensor had very high sensitivity, with the minimum detection limit of 14 μM. Considering that sialic acid locates at the end of cell membrane glycan an play an important role in many biological and pathological processes, these authors further extended the use of PDA fluorescent probes for analysis of SA on the surface of living cells (Wang et al., 2020).

In this review, we summarized the progress of PDA in the field of biological detection and bioimaging. Since the preparation of PDA have a great influence on the performance of the PDA biosensors, we first introduced the latest progress of PDA preparation technology in recent years, including PDA nanomaterials, 3D-PDA, porous PDA, chiral PDA, reversible response PDA and so on. Next, the applications of PDA for biological detection and bioimaging were discussed according to their optical properties. The characteristics and the latest research progress of three types of PDA probes including colorimetry, fluorescence, and Roman were overviewed. In the colorimetric PDA probes, we discussed the source and influencing factors on PDA's absorption and then provided recent progress of colorimetric PDA probes in the biological detection according to the size of targets. In terms of PDA Raman probe and fluorescence probe, we analyzed the origin and influencing factors of Raman scattering and fluorescence, as well as compared the advantages and disadvantages of these two types of probes in biological detection and bioimaging.

PDA probes have great potential in vitro convenient detection such as Point-of-care testing (POCT). PDA can produce distinguished color change from blue to red, which can be easily recognized by human eyes. Therefore, PDA has a great prospect in many fields, such as convenient biological detection, test paper detection, high-throughput detection, among others. However, the signal stability, selectivity and preparation methods of PDA still need further improvement. At present, most PDA bioprobes are in the stage of laboratory investigation. By the cooperation of multi-disciplinary scientists, we believe that the clinical applications of PDA probes in biomedicine will be realized in the future.

Although PDA has great advantages in biomedicine, most of the current applications are carried out in vitro or at the cell level, and its application in real in vivo applications is still limited. PDA can not only be used as a drug carrier, but also has special optical properties such as absorption, fluorescence, and Raman. Therefore, PDA should be very promising in terms of intelligent biomedical materials. We believe that in the near future, PDA smart materials that integrate controlled drug release, specific imaging and detection can truly be used in vivo.

KA and QH designed the framework of the reviews. All authors wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.