Advances in the synthesis of gold nanoclusters (AuNCs) of proteins extracted from nature

2.1.1 BSA

In many parts of the world, cattle are primarily used as a reliable source of human meat and milk, and their main source of nutrition is protein [24]. The accessibility of bovine proteins and their unique physicochemical properties have prompted researchers to explore their function in AuNC synthesis. BSA is the most abundant plasma protein in serum. Its unique physical and chemical properties are widely used in biology and medicine [25]. BSA is a large globular protein composed of numerous acidic and basic amino acids. These include 35 cysteine residues, which have been proven to reduce and combine to form AuNCs under alkaline conditions [26]. BSA was the first protein used to synthesize protein-AuNCs in 2009 [27]. Its spatial structure provides numerous binding sites for gold, and the 35 cysteine residues maintain strong binding to AuNCs via Au-S coordination bonds [28]. Thermogravimetric analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) have shown that the prepared BSA-AuNC core contains 25 gold atoms, which has a typical cluster characteristic and high stability. In addition, the prepared BSA-AuNCs exhibit near-infrared emission characteristics and are very stable under a broad pH range. They can be redispersed in solution and emit strong fluorescence in solid form at room temperature for two months. The pH, temperature, and nature of the reducing agent often have an important influence on the synthesis of protein-AuNCs. Ultraviolet and visible spectrum, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and fluorescence spectra have indicated that AuNCs with fewer than eight atoms grow at pH 7–8, whereas clusters with 25 atoms often grow at pH 11 [29]. BSA-stabilized AuNCs provide a good foundation for the synthesis of protein-AuNCs and have promoted their research and exploration. In previous studies, BSA-stabilized AuNCs have been used to detect mercury ions [30]. The detection is based on the specific quenching of BSA-AuNCs fluorescence by mercury ions. Owing to the high quantum yield (QY) and high fluorescence of BSA-AuNCs, the detection limit for mercury ions is only 0.5 nM, which is considerably lower than the recommended minimum mercury content of 10 nM reported by the U.S. Environmental Protection Agency. However, the selectivity and sensitivity of BSA-stabilised AuNCs depend largely on the pH and ionic strength of the sample solution. To solve this, a novel solution was proposed wherein sodium borohydride and ethylene diamine tetraacetic acid were used to inhibit the binding of metal ions to BSA-AuNCs, thereby slowing down the quenching effect and improving selectivity [31]. Other studies have found that Cu²⁺ can inhibit the fluorescence of BSA-AuNCs by interacting with BSA. Furthermore, dopamine can prevent Cu²⁺ from interfering with BSA-AuNCs fluorescence because of its strong Cu²⁺ binding properties. This function has been used for the rapid detection of dopamine [32]. Because of the crucial role of dopamine in neurotransmission, this method could be developed as a sensitive and rapid diagnostic tool. Gold is a typically inert metal in chemistry, and few anions can react with gold. Interestingly, cyanide can form stable gold-cyanide complexes that have strong covalent bonds, resulting in the fluorescence quenching of BSA-AuNCs when cyanide is present [33]. Therefore, BSA-stabilized AuNCs can be subsequently used to detect cyanide. Meanwhile, cystatin C is a protease inhibitor in the human body, widely present in nucleated cells and body fluids. Because of the correlation between its concentration in serum and some diseases, it is considered a new marker of glomerular filtration rate, which can reflect the health of the kidney. A multifunctional fluorescent sensor based on BSA-AuNCs has been demonstrated for the rapid and sensitive detection of cystatin C and papain [34]. The cysteine protease activity of papain catalyzes the fluorescence quenching of BSA-AuNCs; therefore, the method can be applied to detect papain. However, after the addition of cystatin C, the fluorescence of BSA-AuNCs can be restored; therefore, the method can sensitively determine the concentration of cystatin C in the range of 0.025–2.0 µg/mL with the detection limit of 4.0 ng/mL. Catalase is known to be present in almost all living organisms to break down hydrogen peroxide and provide antioxidant defenses enzymatically. Hydrogen peroxide is the substrate of the reaction. Considering the excellent fluorescence quenching performance of hydrogen peroxide on BSA-AuNCs, a series of quantitative detection methods for enzyme substrates such as glucose and uric acid have been developed [35]. Compared to non-enzymatic detection systems, these methods have the advantages of high sensitivity and specificity, showing considerable diagnostic potential. Quantitative determination methods for hydrogen peroxide based on the principle of fluorescence quenching have emerged extensively. However, the mechanism of hydrogen peroxide-induced AuNC fluorescence quenching remains unclear. Previous studies have suggested that the Au–S bond can be broken by hydrogen peroxide, resulting in the fluorescence quenching of AuNCs [36]. However, other studies have shown that the fluorescence quenching of metal nanoclusters is due to the transfer of protein ligands to the core charge of the metal nanoparticles being hindered [37] (Figure 2).

Figure 2

Schematic diagram of AuNCs synthesized by BSA.

2.1.2 Ovalbumin

In addition to beef and milk, eggs are a main source of protein because they contain essential amino acids for human survival. Eggs are composed of three main components: the egg white, egg yolk, and eggshell. Most of the proteins in eggs are concentrated in egg whites, and there are four main proteins in egg whites that account for approximately 13% of the total mass of egg whites. These include ovalbumin (54%), ovotransferrin (12%), ovoglobulin (11%), and lysozymes (3.4%) [38]. The availability of egg proteins is an advantage for the synthesis of fluorescent nanoclusters. In 2014, egg whites were used as the starting material for preparing fluorescent protein-AuNCs [39]. By mixing egg white with a chloroauric acid aqueous solution at physiological temperature, adjusting the pH to alkaline, and reacting for 12 h, highly fluorescence and water-soluble protein-AuNCs can be obtained. The obtained protein-AuNCs exhibit red photoluminescence [40]. Subsequent studies have optimized the step in a simple and energy-saving manner by changing the reaction conditions to room temperature without stirring. The prepared red-emitting AuNCs have been used as fluorescent sensors for the sensitive detection of hydrogen peroxide [41]. Ovalbumin, the most abundant protein in egg whites, can be used to generate fluorescent protein-AuNCs through efficient synthesis under alkaline conditions. The AuNCs prepared by this method were shown to selectively capture ricin B in very complex samples, including cell lysates, protein mixtures, and powder samples [42]. Each ovalbumin contains six cysteine and ten tyrosine residues, which can reduce and stabilize free gold ions under alkaline conditions [26]. At present, ovalbumin-modified AuNCs can be synthesized using simple and controllable methods. The prepared fluorescent protein-AuNCs exhibit a strong fluorescence emission peak at 650 nm. The prepared clusters have been used as broad-spectrum detectors for real-time detection of tetracycline antibiotics. With an increase in antibiotic concentration, the fluorescence color changes, and the fluorescence intensity at 650 nm decreases [43] (Figure 3).

Figure 3

Schematic diagram of AuNCs synthesized by egg albumin.

2.1.3 Eggshell membrane

Most synthetic preparation methods for AuNCs use aqueous solutions. However, biologically derived proteins often exist in solid form. Therefore, researchers investigated whether solid-state proteins could be used as matrix materials for the synthesis of fluorescent protein-AuNCs. The eggshell membrane is an example of a solid form of protein. It is a double-layered insoluble water film with a natural fiber network naturally generated in the eggshell and is composed of highly crosslinked cysteine-rich proteins with a structure similar to that of keratin, collagen, or elastin. As a good supplement to existing methods, a synthetic method for preparing red fluorescent AuNCs was successfully developed using an eggshell membrane as a reducing agent and stabilizer [44]. Meanwhile, AuNCs with peroxidase-like properties have been prepared using eggshell membranes as templates. First, ESM-AuNCs were combined with GSH to form a complex. As the reaction with glutathione proceeds, the fluorescence intensity of AuNCs decreases, and the catalytic ability increases. Subsequently, the staphylococcal enterotoxin aptamer was immobilized on the ESM membrane. During the detection process, due to the combination of staphylococcal enterotoxin and its aptamer to form a complex, the catalytic site of AuNCs was masked. At this time, the chromogenic enzyme substrate was added, and the color changed. According to the color change, a colorimetric method for rapid detection of staphylococcal enterotoxin was established, which opened up a new method for food safety detection [45] (Figure 4).

Figure 4

Schematic diagram of AuNCs synthesized by Eggshell membrane.

2.1.4 Lysozyme

Lysozyme is an antibacterial enzyme widely found in bacteria, fungi, animals, and plants. Their main function is to destroy bacterial cell walls by hydrolyzing the β-1,4-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan chains. Lysozyme has many excellent properties, such as acid and alkali resistance, strong thermal stability, and excellent antibacterial performance [46]. Egg whites are the main source of lysozyme. However, other sources, including tears, saliva, plasma, mucus, and skin, are also good sources of lysozyme, which can also be found in some plants. Lysozyme was first used in 2010 to replace BSA as a template for synthesizing protein-AuNCs [47]. The synthesized lysozyme-AuNCs exhibited a strong fluorescence emission at approximately 650 nm, with a QY of 5.6%. Transmission electron microscopy (TEM) has confirmed that the formed lysozyme-AuNCs were spherical with a particle size of approximately 1 nm. In the same year, Lin and Tseng conducted similar experiments to synthesize fluorescent AuNCs using lysozyme VI as the template [48]. The excitation and emission wavelengths were 400 and 631 nm, respectively. The molecular weight determined by MALDI-TOF mass spectrometry was 5 kDa. In terms of potential applications, lysozyme VI-AuNCs improved the detection of Hg²⁺ by approximately 330 times compared to its previous value. The detection limits for CH₃Hg⁺ and Hg²⁺ were 4 pM and 3 nM, respectively. Subsequent studies used lysozyme VI under acidic conditions to synthesize highly fluorescent AuNCs [49]. These new lysozyme VI-AuNCs emitted blue fluorescence at 455 nm upon excitation at 380 nm with a considerably high QY of 56%. The authors investigated the differences between the lysozyme VI-AuNCs synthesized at different pH levels by studying their growth mechanisms. At pH 3, the conformation of lysozyme type VI is more compact, therefore, a smaller template for the synthesis of AuNCs. This leads to lysozyme VI-AuNCs with a high QY. Comparatively, high pH values led to the denaturation of lysozyme type VI, resulting in a lower QY. A study demonstrated that functional lysozyme-AuNCs could act as antibacterial agents against drug-resistant bacteria [50]. When excited at 395 nm, the synthesized lysozyme-AuNCs exhibited a strong red fluorescence emission at 640 nm. The QY was 3.6 ± 0.4%, and the TEM image showed that the size of the synthesized lysozyme-AuNCs was approximately 2.3 ± 0.3 nm. The characterization results of inductively coupled plasma mass spectrometry and FTIR showed that almost 100 lysozyme molecules were present in a single AuNC. Furthermore, the authors also observed that lysozyme-AuNCs showed high stability in extremely alkaline and high salt concentration solutions. Subsequently, the prepared lysozyme-AuNCs were used as fluorescent labels and antibacterial sensors for microorganisms. The results showed that the nanoclusters inhibited the growth of drug-resistant bacteria owing to the absence of cysteine residues in lysozyme; therefore, the activity of lysozyme-AuNCs was not affected by Au–S binding. In addition, lysozyme-AuNCs were used as fluorescent probes to detect cyanide anions [51] (Figure 5).

Figure 5

Schematic diagram of AuNCs synthesized by lysozyme.

2.1.5 Trypsin

Trypsin is a serine proteolytic enzyme that acts mainly as a digestive enzyme in vertebrates. It is the most specific protease to achieve the function of hydrolyzing protein by breaking lysine or arginine carboxyl group [52]. In 2011, trypsin was first used to synthesize AuNCs, and fluorescent trypsin-AuNCs with a red emission wavelength of 640 nm were synthesized in an alkaline aqueous solution. The authors found that its photostability was similar to that of cadmium selenide quantum dots and that mercury ions could specifically quench its fluorescence. The authors used this feature to detect mercury ions sensitively and selectively with a detection limit of 50–10 nM [53]. Subsequently, trypsin-AuNCs synthesized by researchers emitted red fluorescence at 645 nm when excited at 360 nm, and TEM images revealed that they had a diameter of approximately 2 nm. MALDI-TOF mass spectrometry results showed that the molecular weight of the trypsin-AuNCs was 5 kDa and that they had a QY of 6.5% [12]. The authors designed a biosensor based on surface plasmon-enhanced cysteamine-gold nanoparticles and trypsin-AuNCs for the detection of heparin and in vivo cancer cell imaging. Heparin-guided energy transfer between trypsin-AuNCs and cysteamine-modified nanogold was used to generate the signal output of the fluorescent biosensor. The linear range of the biosensor was 0.1–4.0 µg/mL, and the heparin detection limit was 0.05 µg/mL. In vivo studies in mice have used folic acid to modify trypsin-AuNCs. This modified folic acid-trypsin-AuNC probe had high specificity for detecting HeLa tumors (Figure 6).

Figure 6

(a) Schematic diagram of the selective detection of heparin for energy transfer between surface plasma-enhanced Cyst-AuNPs and Try-AuNCs. (b) Schematic diagram of folic acid-modified Try-AuNCs for in vivo cancer imaging.

2.1.6 Pepsin

Pepsin was first discovered in the early nineteenth century and was the first enzyme found in animals. Pepsin is a gastric aspartic protease with a molecular weight of approximately 3.4 kDa. The main site of pepsin hydrolysis is the peptide bond formed by the amino groups of aromatic amino acids or acidic amino acids. Pepsin plays an important role in digestion in vertebrates [54]. A team conducted a study and prepared pepsin-AuNCs with blue, green, and red fluorescence emissions at different pH conditions [55]. Among them, Au₂₅NCs (pH = 12) emitted red fluorescence at 670 nm, which was similar to the results of previous studies. Au₁₃NCs (pH = 1) emitted green fluorescence at 510 nm. Au₈NCs and Au₅NCs (pH = 9) emitted blue fluorescence at 480 and 402 nm, respectively. The QY of the red-, green-, and blue-emitting AuNCs were 3.5, 5.0, and 3.7%, respectively. XPS proved that the binding energy of the pepsin-AuNCs increased with decreasing particle size. The authors hypothesized that the red-fluorescent Au₂₅NCs were stabilized by weakly bonded random helical pepsin and that the alkaline synthesis conditions used led to the denaturation of the peptide-protein in the internal space of the enzyme, which resulted in a larger space to bind more gold atoms. At low pH (less than 1), pepsin autolysis occurs, and the smaller internal space produced by the early hydrolysate may act as a template to produce smaller Au₁₃NCs. In the pH range of 3–6, different forms of AuNCs were formed, including gold nanoplates with sizes of hundreds of nanometers (Figure 7).

Figure 7

Schematic diagram of AuNCs synthesized by pepsin under different pH.

An increasing number of animal-derived proteins are being used to prepare protein-AuNCs. For example, researchers have used immunoglobulins to prepare and functionalize AuNCs. The prepared immunoglobulin-AuNCs can emit strong red fluorescence and have high photoluminescence QY [56]. It retains the biological activity of immunoglobulins and enables them to bind to goat anti-human immunoglobulins. The authors developed a simple detection method based on a fluorescence-labeled dot immunoassay that has considerable potential for the development of efficient biosensors. Other researchers used protamine as a template for preparing AuNCs. The authors found that 1-hydroxypyrene could induce the aggregation of AuNCs through hydrogen bonding and electrostatic and hydrophobic interactions, resulting in the enhancement of the photoluminescence of the protamine-AuNCs [57]. Based on this feature, a highly sensitive and selective quantitative strategy for the detection of 1-hydroxypyrene was proposed.

2.2.1 HRP

HRPs, which are present in horseradish plants, have been widely studied for over half a century and are members of the peroxidase family. In 1976, Welinder identified the first complete amino acid sequence of HRP [58]. It contains numerous isozymes. HRP C is the most common member of the plant peroxidase superfamily and is one of the most thoroughly studied members [59]. HRP contains 308 amino acid residues and is rich in tyrosine and cysteine residues. The three-dimensional structure of HRP consists of two domains, the proximal and distal domains, with a heme group located between them. Its amino acid composition and unique three-dimensional structure make it an ideal template for AuNC synthesis. In 2011, for the first time, HRP-AuNCs were greenly synthesized under physiological conditions using HRP as a template [60]. The HRP-AuNCs synthesized by this method not only retain the catalytic function of the enzyme but also exhibit the fluorescence characteristics of AuNCs. The catalytic activity of HRP is well preserved. The addition of hydrogen peroxide quantitatively quenched the fluorescence of the HRP-AuNCs, and it was assessed for hydrogen peroxide detection applications. Recently, an HRP-encapsulated fluorescent bionanomaterial consisting of BSA-stabilized AuNCs and HRP-stabilized AuNCs was reported [61]. The material exhibited a two-step fluorescence quenching effect. Based on this effect, the author designed a device for the detection of hydrogen peroxide. The detection limit was as low as 0.5 nM, which could be detected by the naked eye and had good selectivity. This device can be used to detect other substances and enzymes that produce hydrogen peroxide (Figure 8).

Figure 8

Schematic diagram of AuNCs synthesized by HRP.

2.2.2 Papain

Papain is a cysteine protease, also known as papaya protease, extracted from papaya and has the advantages of low price and easy access. Papain can decompose proteins through amide bond cleavage in acidic, neutral, and alkaline environments and has a wide substrate specificity. It acts on the peptide bonds formed by l-arginine, l-lysine, and l-papain amino acid residue carboxyl groups in the protein. Papain can break down proteins into small fragments; therefore, it is often used in food manufacturing, such as meat processing, or as a stabilizer for brewing [62]. In 2013, papain was first used as a template to synthesize highly fluorescent papain-AuNCs under alkaline conditions [63]. The synthesized papain-AuNCs produced red fluorescence emission at 660 nm and had a QY of 4.3%. Meanwhile, the average particle size of the clusters was 1.2 ± 0.2 nm. In previous studies, 35 cysteine residues in BSA were involved in the formation of BSA-AuNCs. However, papain only contains eight cysteines. The authors speculate that in addition to cysteine residues, other amino acid residues may be involved in the synthesis and growth of AuNCs. Changes in the secondary structure of papain were observed using FTIR and circular dichroism spectroscopy, which confirmed the theory. Compared to BSA, the spectral changes in papain were more obvious, and BSA has more thiol groups than papain. In previous studies, thiol groups have been considered to play an important role in the formation of AuNCs. The fluorescence emission of the synthesized papain-AuNCs is considerably stable at room temperature for over 3 months, making it an ideal biostable fluorescent sensor. The fluorescent probe exhibits high sensitivity and selectivity for the determination of Cu²⁺ by fluorescence quenching, and the Cu²⁺ detection limit is 3 nM. Subsequently, researchers used papain to produce fluorescent papain-AuNCs. d-Penicillamine restored the fluorescence quenching caused by Cu²⁺. To understand this mechanism, a set of quantitative detection methods for d-penicillamine was developed [64]. The papain-AuNCs synthesized produced red fluorescence emission at 639 nm and had an average size of approximately 5.7 nm. Owing to the bonding of Cu²⁺ and papain amino acid residues on the surface of the papain-AuNCs, the nanoclusters exhibited high selectivity and sensitivity to Cu²⁺. In this study, d-penicillamine was added to the papain-AuNC-Cu²⁺ complex, the fluorescence quenched by Cu²⁺ was immediately restored, and the papain-AuNC was found to detect d-penicillamine in real samples with a detection limit of 5.0 mM. Compared with the previous d-penicillamine detection technology [65], this method is not only a green and harmless process. The experimental operation is simple, and the detection is sensitive and selective. In the same year, another researcher invented a one-pot method to generate fluorescent AuNCs using papaya juice as a reducing agent [66]. The prepared papaya juice-AuNCs exhibited green fluorescence emission, and TEM showed that the average size was 6.9 nm. Furthermore, it was found that the fluorescence of papaya juice-AuNCs remained stable for at least 2 months. A papaya juice-AuNC fluorescent probe was used to monitor l-lysine in biological samples, and the detection limit was 6.0 µM. The reaction time was very short, only 120°C reaction for 3 min, thereby achieving the effect of rapid detection. Glyphosate is the most widely used herbicide in the world, and as such, papain was used to prepare AuNCs that showed specific fluorescence quenching for glyphosate and had a QY of 12.4%. Visual or semiquantitative glyphosate detection was performed. In a real sample detection experiment, the sample recovery rate was between 97.3 and 118%, indicating that this detection method has considerable potential for practical applications [67] (Figure 9).

Figure 9

Schematic diagram of AuNCs synthesized by papain.

2.2.3 Soybean protein

Soybeans are a good source of plant protein. Approximately 90% of soybean protein consists of β-conglycinin and glycinin [68]. β-Conglycinin is a trimeric glycoprotein composed of three subunits with a molecular weight of 150–200 kDa. It is rich in content and exhibits strong thermal stability. Soybean globulin is a hexamer with a molecular weight of 300–380 kDa. Researchers have synthesized gold nanomaterials of different shapes and sizes using soybean protein isolate as the precursor [69]. For example, soybean protein isolate induced the formation of gold nanosheets under acidic pH conditions. Meanwhile, at alkaline pH levels, the gold ions captured by the protein are gradually reduced to form AuNCs. The particle size range of soybean protein-AuNCs is 1.3–4.9 nm, and the average particle size is 2.6 nm. The authors believe that the formation of AuNPs during long-term alkaline incubation may be due to the degradation of soybean protein isolate. The degradation of the protein means that its structure cannot couple to larger nanosheets to form nanoparticles. The control experiments conducted in this study have revealed that soybean protein-AuNCs are only stable when synthesized with globular proteins, whereas fibrillin can induce gold ions to form gold nanoparticles but cannot be used as a template for the synthesis of protein-AuNCs. Fluorescence spectroscopy, circular dichroism, and Fourier transform infrared spectroscopy have been used to study and analyze the relationship between the fluorescence changes of soybean protein-AuNCs and the conformational changes in soybean protein ligands during the AuNCs synthesis [13]. According to the fluorescence spectrum analysis, the fluorescence peak intensity before the reaction had taken place was mainly due to the gold core. However, after the reaction had taken place, it was determined that the fluorescence peak intensity was due to a combination of the gold core and the protein-ligand. Circular dichroism and Fourier-transform infrared spectroscopy revealed that the conformation of the soybean protein-ligand changed significantly with increasing reaction time. The structure of the soybean protein changed from ordered to disordered and then to ordered. This proved that the main driving force behind the reaction was to protect the gold nucleus and transfer electrons from the ligand to the gold ion, thereby affecting its fluorescence emission. AuNCs synthesized from soy proteins were used for the detection of bismerthiazol. Bismerthiazol is a fungicide that is used to control bacterial infections in plants, particularly during rice cultivation. Thiadiazole can cause chronic oral toxicity and thyroid damage in rats and may cause similar problems in humans [70]. Currently, high-performance liquid chromatography is used to assess the bismerthiazol content. But there is a need for a quick, simple, and sensitive method with which to improve bismerthiazol sensing [71]. As such, soybean protein-AuNCs were used to develop a method for the rapid and sensitive detection of bismerthiazol in Chinese cabbage [58]. In this experiment, soybean protein was extracted by acid precipitation and alkali extraction before soybean protein-AuNCs were synthesized after freeze-drying. The soybean protein-AuNCs exhibited strong pink fluorescence emission at 600 nm, and their average size was 3 nm. The authors observed that the fluorescence of the clusters was quenched by thiazole. It was postulated that this was due to the presence of two sulfhydryl groups in thiazole that can quench the fluorescence of AuNCs by forming sulfates and metal complexes (Figure 10).

Figure 10

Schematic diagram of AuNCs synthesized by soy protein.

In addition to the aforementioned plant-derived proteins, several researchers have attempted to synthesize AuNCs and apply them in various directions. For example, some researchers have used pea protein to prepare AuNCs with strong red fluorescence, which are stable for up to one month. The pea protein-AuNCs were investigated for their potential in tumor cell imaging [72] (Table 1).