Heparin is a highly sulfated linear polysaccharide, mainly composed of different sulfated disaccharide units such as iduronic acid/glucuronic acid, and glucosamine residues [1], [2], [3], [4]. Heparin has been used clinically as an anticoagulant drug for more than 80 years [5], [6]. Because excessive heparin can lead to side effects such as bleeding and thrombocytopenia [7], [8], strict dosage control is required when using heparin [9]. For example, during cardiopulmonary surgery and emergency deep vein thrombosis (DVT), the concentration of heparin needs to be controlled at 2–8 U mL−1 (0.8–3.2 µM); after DVT surgery and long-term anticoagulation therapy, it is necessary to control the therapeutic level of heparin in patients to be 0.2–2 U mL−1 (0.08–0.8 µM) [10].

Many fluorescent probes based on organic dyes, cationic polymers, dye-labeled peptides and MOF nanomaterial etc have been developed for heparin detection [11]. Especially, the Singh group developed various fluorescence turn-on probes for the specific and sensitive detection of heparin, which exhibited good performance in complex biological media of both human serum and plasma matrix [12], [13], [14], [15], [16], [17], [18]. Most of these probes carry positively charged groups that could electrostatically interact with negatively charged heparin, inducing the changes in fluorescent signals. For example, with a fluorescent scaffold of 1,3,5-triphenylethylbenzene and heparin receptor of ammonium containing boronate ester [10]; with rhodamine B and positively charged peptide RNRHTHLRTRPRK [19]; or with cationic fluorescent perylene diimide probe [20] all have been designed as the aggregation caused quenching (ACQ) probes for the detection of heparin. Nevertheless, ACQ effect usually suffers from fluorescence self-quenching and decreased fluorescence efficiency [21], [22]. Recently, to avoid the ACQ defect, several “turn on”, especially aggregation-induced emission (AIE) fluorescent probes have been developed [23], [24]. For fluorescence sensing, the “turn on” probes, which could effectively eliminate the false fluorescent signals induced by uncertain factors, have been considered to be more reliable than the “turn off” ones [21], [22], [25].

AIE has been considered to be a promising strategy for the development of “turn on” fluorescent probes for heparin detection [25], [26]. The AIE fluorophores are almost non-fluorescent in the solution state but emit strong fluorescence after analytes induced aggregation [27], [28], [29], [30]. For example, through combining leucine and tetraphenylethene (TPE), the Wei group developed the pH-sensitive AIE probe named TPE-Leu, which could achieve the sensitive detection of urease and acetylcholinesterase [31]. Meanwhile, inspired by the fact that heparin shows a high binding affinity toward positively charged peptides units composed of suitable amino acid sequence, considerable efforts have been devoted to introduce the heparin-specific binding peptide in the luminescent probes [23], [24], [32].

Especially, the linear peptide AG73, which contains 12 amino acid residues derived from the G domain of the laminin α1 chain, has been demonstrated to be able to bind specifically to heparin [25]. It is worth mentioning that AG73 not only has the ability to bind to heparin, but also has other functions, such as enhancing cell attachment and spreading [33]. Through combining AG73 and pyrene fluorophore, the Lee group developed the sensor Py12, which could detect heparin with nanomolar sensitivity [34]. Through modulating the photoluminescence of Tb3+ with ssDNA, the Wei group developed Tb3+-ssDNA-AG73 time-resolved luminescence system for heparin detection [35]. Meanwhile, the Wei group also developed a series of AG73-modified 2D metal–organic framework (MOF) nanosheets, which was applied for quantitative monitoring the elimination of heparin in live rats [36].

The fluorophore TPE is the classic AIE fluorophore. Through combining AG73 and TPE, the Wei group developed the “turn on” fluorescent probe TPE-1 for heparin detection [25]. The detection mechanism was ascribed to the aggregation of TPE-1 onto the surface of heparin via AG73, which could sensitively and selectively lead to “turn-on” the AIE fluorescence of the TPE moiety (Fig. 1). As reported, TPE-1 probe had excellent AIE luminescence response to heparin, including the low detection limit, high selectivity, and good linear range. Nevertheless, previous studies have indicated that linear peptides may suffer from poor proteolytic stabilities and short half-lives in human serum [37], [38], [39], [40], [41], [42], [43]. Especially, the AG73 with the amino acid sequence of RKRLQVQLSIRT is rich in arginine (R) residues, which tends to be degraded by various proteases [44]. Moreover, the TPE moiety containing four hydrophobic benzene rings exhibited extremely poor solubility. Therefore, although peptide based AIE probes have been considered as an excellent fluorescent probe for heparin detection, some significant deficiencies related to its low proteolytic stability and poor solubility should be addressed to facilitate their widespread applications. Nevertheless, to the best of our knowledge, little effort has been devoted to structural optimization of TPE-1.

In order to improve both the proteolytic stability and hydrophility of TPE-1, the structural optimizations were conducted on TPE-1, and the derived fluorescent probes were efficiently synthesized and evaluated in this study. Recently, d-type peptides have been considered as novel molecular skeletons due to their striking enzymatic hydrolysis-resistant property and low immunogenicity [45], [46], [47]. Thus, we performed the first d-type amino acid substitution for AG73 peptide. Meanwhile, to improve the solubility of probes, the solubilizing tags were incorporated into different probes [48], [49]. Subsequently, with seven TPE-1 derived probes in hand, the stability, water solubility, as well as the heparin detection potential were systematically evaluated. Especially, to achieve real-time monitoring of peptide integrities, the novel Abz/Dnp-based “turn on” probes that employ the internally quenched fluorescent (IQF) mechanism [50], [51] was synthesized and applied in both protease and serum environments.

The l-type to d-type amino acid residue substitution, combined with the incorporation of solubilizing tag, afforded the derived probe XH-6. It was found that XH-6 exhibited extremely higher proteolytic stability and solubility than those of TPE-1. Meanwhile, XH-6 possessed comparable heparin detection efficiency as that of TPE-1. Collectively, this study not only established practical strategies to improve both the water solubility and proteolytic stability of “turn on” fluorescent probes of heparin, but also provided valuable references for the subsequent development of hydrolysis-resistant d-type peptides based fluorescent probes.