Silk fibroin-based hemostatic powders with instant and robust adhesion performance for sutureless sealing of gastrointestinal defects.

Powder-based hemostatic technology has offered unprecedented opportunities in surgical sealing and repair of irregularly shaped and noncompressible wounds. Despite their routine use, existing clinical hemostatic powders are challenged either by poor mechanical properties or inadequate adhesion to bleeding tissues in biological environments. Here, inspired by the mussel foot proteins' fusion assembly strategy, a novel silk fibroin-based hemostatic powder (named as SF/PEG/TA) with instant and robust adhesion performance is developed. Upon absorbing interfacial liquids, the SF/PEG/TA powders rapidly swell into micro-gels and subsequently contact with each other to transform into a macroscopically homogeneous hydrogel in situ, strengthening its interfacial bonding with various substrates in fluidic environments. The in vitro and in vivo results show that the SF/PEG/TA powder possesses ease of use, good biocompatibility, strong antibacterial activities, and effective blood clotting abilities. The superior hemostatic sealing capability of the SF/PEG/TA powder is demonstrated in the rat liver, heart, and gastrointestinal injury models. Moreover, in vivo investigation of rat skin incision and gastrointestinal perforation models validates that the SF/PEG/TA powder promotes wound healing and tissue regeneration. Taken together, compared to existing clinical hemostatic powders, the proposed SF/PEG/TA powder with superior wound treatment capabilities has high potential for clinical hemostasis and emergency rescue.

An Artificial Liquid-Liquid Phase Separation-Driven Silk Fibroin-Based Adhesive for Rapid Hemostasis and Wound Sealing.

The powerful adhesion systems of marine organisms have inspired the development of artificial protein-based bioadhesives. However, achieving robust wet adhesion using artificial bioadhesives remains technically challenging because the key element of liquid-liquid phase separation (LLPS)-driven complex coacervation in natural adhesion systems is often ignored. In this study, mimicking the complex coacervation phenomenon of marine organisms, an artificial protein-based adhesive hydrogel (SFG hydrogel) was developed by adopting the LLPS-mediated coacervation of the natural protein silk fibroin (SF) and the anionic surfactant sodium dodecylbenzene sulfonate (SDBS). The assembled SF/SDBS complex coacervate enabled precise spatial positioning and easy self-adjustable deposition on irregular substrate surfaces, allowing for tight contact. Spontaneous liquid-to-solid maturation promoted the phase transition of the SF/SDBS complex coacervate to form the SFG hydrogel in situ, enhancing its bulk cohesiveness and interfacial adhesion. The formed SFG hydrogel exhibited intrinsic advantages as a new type of artificial protein-based adhesive, including good biocompatibility, robust wet adhesion, rapid blood-clotting capacity, and easy operation. In vitro and in vivo experiments demonstrated that the SFG hydrogel not only achieved instant and effective hemostatic sealing of tissue injuries but also promoted wound healing and tissue regeneration, thus advancing its clinical applications. STATEMENT OF SIGNIFICANCE: Marine mussels utilize the liquid-liquid phase separation (LLPS) strategy to induce the supramolecular assembly of mussel foot proteins, which plays a critical role in strong underwater adhesion of mussel foot proteins. Herein, an artificial protein-based adhesive hydrogel (named SFG hydrogel) was reported by adopting the LLPS-mediated coacervation of natural protein silk fibroin (SF) and anionic surfactant sodium dodecylbenzene sulfonate (SDBS). The assembled SFG hydrogel enabled the precise spatial positioning and easy self-adjustable deposition on substrate surfaces with irregularities, allowing tight interfacial adhesion and cohesiveness. The SFG hydrogel not only achieved instant and effective hemostatic sealing of tissue injuries but also promoted wound healing and tissue regeneration, exhibiting intrinsic advantages as a new type of artificial protein-based bioadhesives.

A disulfide molecule-vancomycin nanodrug delivery system efficiently eradicates intracellular bacteria.

Intracellular bacteria often lead to chronic and recurrent infections; however, most of the known antibiotics have poor efficacy against intracellular bacteria due to their poor cell membrane penetration efficiency into the cytosol. Here, a thiol-mediated nanodrug delivery system, named Van-DM NPs, was developed to improve vancomycin's penetration efficiency and intracellular antibacterial activities. Van-DM NPs were prepared through self-assembly of vancomycin (Van) and the disulfide molecule (DM) in NaOH buffer solution. On the one hand, the disulfide exchange reaction between Van-DM NPs and the bacterial surface enhances vancomycin accumulation in bacteria, increasing the local concentration of vancomycin. On the other hand, the disulfide exchange reaction between Van-DM NPs and the mammalian cell membrane triggered the translocation of Van-DM NPs across the mammalian cell membrane into the cell cytosol. These dual mechanisms promote antibacterial activities of vancomycin against both extracellular and intracellular bacteria S. aureus. Furthermore, in an intravenous S. aureus infection mouse model, Van-DM NPs exhibited high antibacterial capability and efficiently reduced the bacterial load in liver and spleen, where intracellular bacteria tend to reside. Altogether, the reported Van-DM NPs would be highly promising against intracellular pathogenic infections.

Natural polymer-based bioadhesives as hemostatic platforms for wound healing.

Medical adhesives are advanced but challenging alternatives to wound closure and repair, especially in mitigating uncontrolled hemorrhage. Ideal hemostatic adhesives need to meet good biocompatibility and biodegradability, adequate mechanical strength, and strong tissue adhesion functionality under wet and dynamic conditions. Considering these requirements, natural polymers such as polysaccharide, protein and DNA, attract great attention as candidates for making bioadhesives because of their distinctive physicochemical performances and biological properties. This review systematically summarizes the advances of bioadhesives based on natural polysaccharide, protein and DNA. Various physical and chemical cross-linking strategies have been introduced for adhesive synthesis and their hemostatic applications are introduced from the aspect of versatility. Furthermore, the possible challenges and future opportunities of bioadhesives are discussed, providing insights into the development of high-performance hemostatic materials.

Silk Fibroin-Based Tough Hydrogels with Strong Underwater Adhesion for Fast Hemostasis and Wound Sealing.

Rapid and strong adhesion of hydrogel adhesives is required for instant wound closure and hemostasis. However, in situ hydrogel formation and sufficient adhesion at target tissue sites in biological environments are severely compromised by the presence of blood and body fluids. In this work, an underwater adhesive hydrogel (named SHCa) is fabricated with rapid in situ gelation, enhanced mechanical toughness, and robust underwater adhesion. The SHCa can undergo rapid UV irradiation-induced gelation under water within 5 s and adhere firmly to underwater surfaces for 6 months. The synergistic effects of crystalline ß-sheet structures and dynamic energy-dissipating mechanisms enhance the mechanical toughness and cohesion, supporting the balance between adhesion and cohesion in wet environments. Importantly, the SHCa can achieve rapid in situ gelation and robust underwater adhesion at various tissue surfaces in highly dynamic fluid environments, substantially outperforming the commercially available tissue adhesives. The lap shear adhesion strength and wound closure strength of SHCa on blood-covered substrates are 7.24 and 12.68 times higher than those of cyanoacrylate glue, respectively. Its fast hemostasis and wound sealing performance are further demonstrated in in vivo animal models. The proposed hydrogel with strong underwater adhesion provides an effective tool for fast wound closure and hemostasis.

Biomimetic multifunctional hybrid sponge via enzymatic cross-linking to accelerate infected burn wound healing.

Preparing sponge dressings with stable wet adhesion remains difficult in wound repair, especially in burn wounds with bleeding and large amounts of exudate. In this work, a multifunctional hybrid sponge dressing (DHGT+PHMB+TiO2NPs) with good wet adhesion was developed by combining biomimetic and enzymatic cross-linking reactions. The sponge dressing matrix (DHGT) was prepared by tyrosinase-catalyzed cross-linking of dopamine-modified hyaluronic acid (DOPA-HA) and gelatin. The multifunctional hybrid sponge dressing was obtained by loading polyhexamethylene biguanide (PHMB) and titanium dioxide nanoparticles (TiO2NPs) onto the DHGT matrix. The newly developed sponge dressing exhibited high mechanical properties, good wet adhesion, antibacterial activity, reactive oxygen species (ROS) scavenging, biocompatibility, and excellent hemostasis ability. In vivo studies showed that the multifunctional hybrid sponge dressing could significantly accelerate the healing of infected full-thickness burn wounds by inhibiting bacterial growth, accelerating skin tissue reepithelialization, collagen deposition, and angiogenesis, as well as regulating the expression of inflammatory factors and cytokines.

Advanced bioactive hydrogels for the treatment of myocardial infarction.

Myocardial infarction (MI) is the leading cause of cardiovascular disease-related deaths. Local ischemia and cardiomyocyte death lead to a series of pathological remodeling events of the infarcted extracellular matrix (ECM) that are significantly different from normal cardiac tissues. These pathological characteristics have inspired the development of microenvironment-responsive therapeutic strategies for MI. Bioactive hydrogels attract great attention because of their unique physicochemical features to induce specific biological responses upon interacting with cells, ECMs, and signal molecules. They exhibit physicochemical-responsive activities to regulate interactions between hydrogels and the biological system. Many bioactive hydrogels with intelligent properties have been used to repair infarcted myocardium and restore the cardiac function after MI. Current research studies ensure that bioactive hydrogels have a high clinical importance and application prospects. This review summarizes the advances of bioactive hydrogels in the field of on-demand treatment of MI and evaluates their key physicochemical characteristics. A range of bioactive hydrogels and their biomedical applications are discussed in repairing injured hearts and restoring cardiac functions, providing insights into the development of intelligent therapeutic approaches for MI.

A mussel-inspired supramolecular hydrogel with robust tissue anchor for rapid hemostasis of arterial and visceral bleedings.

In recent years, the developed hemostatic technologies are still difficult to be applied to the hemostasis of massive arterial and visceral hemorrhage, owing to their weak hemostatic function, inferior wet tissue adhesion, and low mechanical properties. Herein, a mussel-inspired supramolecular interaction-cross-linked hydrogel with robust mechanical property (308.47 ± 29.20 kPa) and excellent hemostatic efficiency (96.5% ± 2.1%) was constructed as a hemostatic sealant. Typically, we combined chitosan (CS) with silk fibroin (SF) by cross-linking them through tannic acid (TA) to maintain the structural stability of the hydrogel, especially for wet tissue adhesion ability (shear adhesive strength = 29.66 ± 0.36 kPa). Compared with other materials reported previously, the obtained CS/TA/SF hydrogel yielded a lower amount of blood loss and shorter time to hemostasis in various arterial and visceral bleeding models, which could be ascribed to the synergistic effect of wound closure under wet state as well as intrinsic hemostatic activity of CS. As a superior hemostatic sealant, the unique hydrogel proposed in this work can be exploited to offer significant advantages in the acute wound and massive hemorrhage with the restrictive access of therapeutic moieties.

Biologically Responsive Plasmonic Assemblies for Second Near-Infrared Window Photoacoustic Imaging-Guided Concurrent Chemo-Immunotherapy.

We developed dual biologically responsive nanogapped gold nanoparticle vesicles loaded with immune inhibitor and carrying an anticancer polymeric prodrug for synergistic concurrent chemo-immunotherapy against primary and metastatic tumors, along with guided cargo release by photoacoustic (PA) imaging in the second near-infrared (NIR-II) window. The responsive vesicle was prepared by self-assembly of nanogapped gold nanoparticles (AuNNPs) grafted with poly(ethylene glycol) (PEG) and dual pH/GSH-responsive polyprodug poly(SN38-co-4-vinylpyridine) (termed AuNNP@PEG/PSN38VP), showing intense PA signal in the NIR-II window. The effect of the rigidity of hydrophobic polymer PSN38VP on the assembled structures and the formation mechanism of AuNNP@SN38 Ve were elucidated by computational simulations. The immune inhibitor BLZ-945 was encapsulated into the vesicles, resulting in pH-responsive release of BLZ-945 for targeted immunotherapy, followed by the dissociation of the vesicles into single AuNNP@PEG/PSN38VP. The hydrophilic AuNNP@PEG/PSN38VP nanoparticles could penetrate deep into the tumor tissues and release the anticancer drug SN38 under the reductive environment. A PA signal in the NIR-II window in the deep tumor region was obtained. The BLZ-945-loaded vesicle enabled enhanced PA imaging-guided concurrent chemo-immunotherapy efficacy, inhibiting the growth of both primary tumors and metastatic tumors.

High-Strength and High-Toughness Silk Fibroin Hydrogels: A Strategy Using Dynamic Host-Guest Interactions.

Natural polymer-based hydrogels attract great attention because of their inherent biocompatibility and controllable biodegradability. However, the broad applications of these hydrogels require a combination of high mechanical strength, high toughness, fatigue resistance, as well as self-healing. The integration of this combination into one natural polymer-based hydrogel remains challenging. Here, a molecular design strategy was proposed to fabricate mechanically robust silk fibroin-based hydrogels using host-guest interactions. Silk fibroin molecules was chemically modified with cholesterol (Chol, guest) or ß-cyclodextrin (ß-CD, host), and host-guest interaction between Chol and ß-CD moieties drove the supramolecular assemblies of hydrogels. The dissociation/reassociation behavior of host-guest complexation, serving as sacrificial bonds, endowed hydrogels with effective energy dissipation and rapid self-healing ability. The prepared silk fibroin-based hydrogels exhibited high mechanical strength, high toughness, and remarkable fatigue resistance, superior to conventional silk fibroin hydrogels. Moreover, due to reversible host-guest interactions, hydrogels achieved facile functional recovery after damage without any external stimuli. This design strategy provides an avenue to develop natural polymer-based materials with robust mechanical properties, thus broadening current hydrogel applications.

Water-Based Black Phosphorus Hybrid Nanosheets as a Moldable Platform for Wound Healing Applications.

Black phosphorus (BP) nanosheets with unique biocompatibility and superior optical performance have attracted enormous attention in material science. However, their instability and poor solution-processability severely limit their clinical applications. In this work, we demonstrate the use of silk fibroin (SF) as an exfoliating agent to produce thin-layer BP nanosheets with long-term stability and facile solution-processability. Presence of SF prevents rapid oxidation and degradation of the resultant BP nanosheets, enhancing their performance in physiological environment. The SF-modified BP nanosheets exhibit subtle solution-processability and are fabricated into various BP-based material formats. As superior photothermal agents, BP-based wound dressings effectively prevent bacterial infection and promote wound repair. Therefore, this work opens new avenues for unlocking current challenges of BP nanosheet applications for practical biomedical purposes.

A black phosphorus nanosheet-based siRNA delivery system for synergistic photothermal and gene therapy.

Gene therapy with small interfering RNA (siRNA) has been proved to be a promising technology to treat various diseases by hampering the production of target proteins. However, developing a delivery system that has high efficiency in transporting siRNA without obvious side effects remains a challenge. Herein, we designed a new survivin siRNA delivery system based on polyethyleneimine functionalized black phosphorus (BP) nanosheets which could suppress tumor growth by silencing survivin expression. Combined with the photothermal properties of the BP nanosheets, the presented delivery system shows excellent therapy efficiency for tumors. Therefore, the BP-based delivery system would be a promising tool for future clinical applications.

Silk fibroin-assisted exfoliation and functionalization of transition metal dichalcogenide nanosheets for antibacterial wound dressings.

Two-dimensional transition metal dichalcogenides (TMDs) have attracted rapidly increasing attention due to their fascinating properties and potential applications. However, scalable and cost-effective methods to produce thin-layer TMD nanosheets and their functional composites with environmental benignity are still limited. Herein, we develop a facile and environmentally friendly method for the scalable production of thin-layer TMD nanosheets in an aqueous medium by using silk fibroin, a natural and abundant biopolymer, as the exfoliating agent. Specifically, carboxyl-modified silk fibroin significantly improves the exfoliation efficiency, achieving the high-yield production of thin-layer MoSe2 nanosheets with good solution stabilization and excellent biocompatibility. Strong binding interactions endow the resultant MoSe2 nanosheets with unprecedentedly high concentrations. By virtue of the solution-processability of silk fibroin, MoSe2 hybrid nanosheets are readily fabricated into macroscopic freestanding films. Furthermore, due to the superior peroxidase-like activity of MoSe2 nanosheets, MoSe2-based films show rapid and effective wound disinfection and healing efficacy with the use of low-dose H2O2in vivo, avoiding the side effects of high-dose H2O2 in traditional medical therapy. This work may offer new opportunities to apply two-dimensional TMD nanosheets for clinical applications.

Monodisperse phase transfer and surface bioengineering of metal nanoparticles via a silk fibroin protein corona.

Uniform hydrophobic nanoparticles synthesized in nonpolar solvents possess excellent physio-chemical properties, showing great potential in biomedical applications. However, the presence of hydrophobic ligands on their surfaces limits their use under physiological conditions. Inspired by protein coronas present at the nano-bio interface, here we report a facile and universal method for phase transfer and surface bioengineering of hydrophobic nanoparticles using ß-sheet-rich silk fibroin, a FDA-approved natural protein. Due to its amphiphilicity and high mechanical stiffness, the ß-sheet-rich silk fibroin not only readily drags nanoparticles from an organic phase into aqueous media but also endows them with excellent monodispersity and long-term stability. The silk fibroin-coated nanoparticles can retain the magnetic and optical properties of the original nanoparticles, acting effectively as probes for biomedical imaging and biosensing. Furthermore, hydrophobic drugs can be easily adsorbed onto the protein coating via hydrophobic interaction, allowing the construction of promising theranostic nanoagents. Given these unique features, the strategy developed here possesses great promise in facilitating biomedical applications of hydrophobic nanomaterials.

Silk scaffolds with tunable mechanical capability for cell differentiation.

Bombyx mori silk fibroin is a promising biomaterial for tissue regeneration and is usually considered an "inert" material with respect to actively regulating cell differentiation due to few specific cell signaling peptide domains in the primary sequence and the generally stiffer mechanical properties due to crystalline content formed in processing. In the present study, silk fibroin porous 3D scaffolds with nanostructures and tunable stiffness were generated via a silk fibroin nanofiber-assisted lyophilization process. The silk fibroin nanofibers with high ß-sheet content were added into the silk fibroin solutions to modulate the self-assembly, and to directly induce water-insoluble scaffold formation after lyophilization. Unlike previously reported silk fibroin scaffold formation processes, these new scaffolds had lower overall ß-sheet content and softer mechanical properties for improved cell compatibility. The scaffold stiffness could be further tuned to match soft tissue mechanical properties, which resulted in different differentiation outcomes with rat bone marrow-derived mesenchymal stem cells toward myogenic and endothelial cells, respectively. Therefore, these silk fibroin scaffolds regulate cell differentiation outcomes due to their mechanical features.

Osteoinductive-nanoscaled silk/HA composite scaffolds for bone tissue engineering application.

Osteoinductive silk/hydroxyapatite (HA) composite scaffolds for bone regeneration were prepared by combining silk with HA/silk core-shell nanoparticles. The HA/silk nanoparticles were directly dispersed in silk solution to form uniform silk/HA blend and then composite scaffolds after a freeze-drying process. The HA/silk nanoparticles uniformly distributed in silk scaffolds at nanometer scale at varying HA content up to 40%, and substantially improved the compressive strength of the scaffolds produced. Rat bone mesenchymal stem cells (rBMSCs) were cultured in these scaffolds and cell proliferation was analyzed by confocal microscopy and DNA assay. Gene expression and biochemical assays were employed to study the influence of increasing HA/silk nanoparticles on in vitro osteogenic differentiation of rBMSCs. Increasing HA/silk nanoparticles inside silk scaffolds improved the growth and osteogenic capability of rBMSCs in the absence of osteogenic growth factors, and also significantly increased the calcium and collagen I deposition. In addition, compared to silk/HA composite scaffolds containing HA aggregates, the scaffolds loaded with HA/silk nanoparticles showed remarkably higher stiffness and better osteogenic property at same HA content, implying a preferable microenvironment for rBMSCs. These results suggest that the osteogenic property as well as mechanical property of silk/HA scaffolds could be further improved through fabricating their structure and topography at nanometer scale, providing more suitable systems for bone regeneration.

Silk Nanofiber Hydrogels with Tunable Modulus to Regulate Nerve Stem Cell Fate.

Reconstruction of damaged nerves remains a significant unmet challenge in clinical medicine. To foster improvements, the control of neural stem cell (NSC) behaviors, including migration, proliferation and differentiation are critical factors to consider. Topographical and mechanical stimulation based on the control of biomaterial features is a promising approach, which are usually studied separately. The synergy between topography and mechanical rigidity could offer new insights into the control of neural cell fate if they could be utilized concurrently in studies. To achieve this need, silk fibroin self-assembled nanofibers with a beta-sheet-enriched structure are formed into hydrogels. Stiffness is tuned using different annealing processes to enable mechanical control without impacting the nanofiber topography. Compared with nonannealed nanofibers, NSCs on methanol annealed nanofibers with stiffness similar to nerve tissues differentiate into neurons with the restraint of glial differentiation, without the influence of specific differentiation biochemical factors. These results demonstrate that combining topographic and mechanical cues provides the control of nerve cell behaviors, with potential for neurogenerative repair strategies.

Reversible hydrogel-solution system of silk with high beta-sheet content.

Silkworm silk has been widely used as a textile fiber, as biomaterials and in optically functional materials due to its extraordinary properties. The ß-sheet-rich natural nanofiber units of about 10-50 nm in diameter are often considered the origin of these properties, yet it remains unclear how silk self-assembles into these hierarchical structures. A new system composed of ß-sheet-rich silk nanofibers about 10-20 nm in diameter is reported here, where these nanofibers formed into "flowing hydrogels" at 0.5-2% solutions and could be transformed back into the solution state at lower concentrations, even with a high ß-sheet content. This is in contrast with other silk processed materials, where significant ß-sheet content negates reversibility between solution and solid states. These fibers are formed by regulating the self-assembly process of silk in aqueous solution, which changes the distribution of negative charges while still supporting ß-sheet formation in the structures. Mechanistically, there appears to be a shift toward negative charges along the outside of the silk nanofibers in our present study, resulting in a higher zeta potential (above -50 mV) than previous silk materials which tend to be below -30 mV. The higher negative charge on silk nanofibers resulted in electrostatic repulsion strong enough to negate further assembly of the nanofibers. Changing silk concentration changed the balance between hydrophobic interactions and electrostatic repulsion of ß-sheet-rich silk nanofibers, resulting in reversible hydrogel-solution transitions. Furthermore, the silk nanofibers could be disassembled into shorter fibers and even nanoparticles upon ultrasonic treatment following the transition from hydrogel to solution due to the increased dispersion of hydrophobic smaller particles, without the loss of ß-sheet content, and with retention of the ability to transition between hydrogel and solution states through reversion to longer nanofibers during self-assembly. These reversible solution-hydrogel transitions were tunable with ultrasonic intensity, time, or temperature.

Nanoscale Control of Silks for Nanofibrous Scaffold Formation with Improved Porous Structure.

Silk-based porous scaffolds have been used extensively in tissue engineering because of their excellent biocompatibility, tunable biodegradability and robust mechanical properties. Although many silk-based scaffolds have been prepared through freeze-drying, a challenge remains to effectively control porous structures during this process. In the present study silk fibroin with different nanostructures were self-assembled in aqueous solution by repeated drying-dissolving process and then used to improve porous structure formation in lyophilization process. Viscosity, secondary structures and water interactions were also studied to exclude their influence on the formation and control of porous structures. Following nanofiber formation in aqueous solution, silk scaffolds with improved porous structure were directly formed after lyophilization and then stabilized with water or methanol annealing treatments. Compared to silk scaffolds derived from fresh solution, the nanofibrous scaffolds showed significantly better cell compatibility in vitro. Therefore, this nanoscale control of silk offers feasible way to regulate the matrix features including porous structure and nanostructure, which are important in regulating cell and tissue outcomes in tissue engineering and regeneration, and then achieve silk-based scaffolds with improved properties.

Molecular detection of Penicillium griseofulvum as the coastal pollution indicator.

Molecular methods were carried out to detect Penicillium griseofulvum, a dominant species related to heavy metal pollution, which was screened from marine contaminated sediments. Based on differences in internal transcribed spacer (ITS) sequences of Penicillium genus and specific isoamyl alcohol oxidase (IAO) sequences, species-specific primers AS1/RS4 and IAO1/IAO2 of Penicillium griseofulvum were designed and synthesized which were then employed in optimized PCR systems. The detection sensitivities were compared through ordinary PCR and nested-PCR using two pairs of primers, respectively. Both primer pairs could exclusively amplify destined DNA fragment from contaminated environmental samples in our researches. As for primers AS1/RS4, the detection sensitivity for spores (pure spore DNA) could be 10 fg/µl and 10 spores, respectively, and the detection sensitivity for the sediments was 10² spores/0.25 g sediments. While the detection sensitivity of IAO1/IAO2 primers was lower than that of AS1/RS4. Despite the difference in detection sensitivity, it is feasible that the species-specific primers could be used as probes for the detection of environmental pollution dominant species, Penicillium griseofulvum, since the frequency of occurrence and amount of this strain could preferably indicate the pollution degree.