Background

Recent biomedical applications have revealed the significant roles of nanomaterials in the developments of nanoscience and nanotechnology [1,2,3,4,5,24,25,26,27,5) [79]. It was revealed from the results that cancer cells ingested these molecules to a much larger extent than normal cells [80]. It has been reported [81] that gold nanoparticles are easily penetrable to more mature cells such as granulocytes and lymphocytes which are part of hematopoietic system. Similarly, AuNCs can also be applicable in selective labeling, imaging, and target drug delivery in the hematopoietic system and related cancers such as chronic myeloid leukemia.

Fig. 5
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Labelling of normal and cancer cells with AuNCs and quantum dots (QDs) [79]

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Aldeek et al. designed fluorescent polyethylene glycol- and zwitterion-functionalized gold nanoclusters by using bidentate ligands made of lipoic acid anchoring groups conjoined with either a poly(ethylene glycol) short chain or a zwitterion group [82]. To determine the role of these nanoclusters in biology, various tests were performed like pH-dependent stability and stability in the presence of excess salt. The hypothesis given by the author reveals that these tests are in relevancy to the use of these AuNCs as fluorescent platforms for imaging and sensing in biology. It is further described in the report that various biological abnormalities are related to pH and thus can provide an indication for progression of several diseases such as cancer metastasis, chronic fatigue, and depression [83, 84]. These clusters are also thought to manage the physical behavior of proteins and nucleic acids [85,86,87]. One of the additional benefit of these clusters is their use in in vivo (deep-tissue) imaging. Chen et al. developed a pH-dependent amphiphilic polymeric system containing luminescent AuNCs that were found to be photostable and biocompatible in the form of nanocomposite for diagnostic activities which includes detection and therapy of folate over-expressing cancerous cells [88]. Luminescent AuNCs were restrained with amphiphilic copolymer (poly(DBAM-co-NASco-HEMA) (PDNH)) to form L-nAuNCs/FA-modified PDNH (or L-AuNCs/FA-PDNH) nanocomposite. Furthermore, hydrophobic drug paclitaxel was assembled with L-AuNCs/FA-PDNH and thus can be utilized for both imaging and treatment of cancer (Fig. 6).

Fig. 6
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Fabrication of L-AuNCs/FA-PDNH nanocomposite for imaging and therapy [88]

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Polycations-functionalized water-soluble polyethyleneimine gold nanoclusters (PEI-AuNCs) were designed and synthesized for appropriate and safe gene therapy applications along with cell imaging [89]. Due to the enthralling optical properties of PEI-AuNCs, these clusters are reported as a promising candidate for bioimaging, which was confirmed by incubating cancer cell lines (HepG2) with PEI-AuNCs and showed remarkable photoluminescence and the cells giving strong intense red fuorescence. Gold nanoclusters protected by ovalbumin (fluorescent probe) linked with folic acid (targeting ligand) (FA-Ova-AuNCs) and a homopolymer N-acryloxysuccinimide as the linker is being developed by Qiao et al. and was utilized for the detection of cancer through cancer cell imaging (Fig. 7). As folic acid receptors are over-expressed in HeLa cells, it is thought that Hela cells would ingest FA-Ova-AuNCs. In this work, specific staining of HeLa cells by FA-Ova-AuNCs has been demonstrated [90].

Fig. 7
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Schematic of the formation of FA-Ova-AuNCs for cancer cell imaging [90]

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For the application in detection, highly phosphorescent molecular gold(I) cluster in a macroporous polymer film has been designed and synthesized for the detection of cyanide through colorimetric detection technique. The gold nanoclusters can be used to detect cyanide ions in red wine, coffee, juice, and soil. As cyanide is extremely toxic and hazardous and may lead to death [91], there was a need to find highly selective, sensitive, and cost-effective sensors that can help determine the cyanide levels in the environment, water, and food [92]. So, this gold nanocluster may act as benediction which may help to save a number of lives [93].

Biomacromolecule-Conjugated AuNCs

Biomacromolecules with thiol groups have also been applied as commonly used ligands to prepare AuNCs in different biomedical applications. Recently, transferrin (Tf)-functionalized gold nanoclusters (Tf-AuNCs)/graphene oxide (GO) nanocomposite (Tf-AuNCs/GO) was manufactured as a turn-on near-infrared (NIR) fluorescent probe which can be utilized for bioimaging of cancer cells and small animals [94]. The ability of NIR fluorescent probe for imaging Tf receptor (TfR) on cancer cells was evaluated using two different cancer cell lines, i.e., Hela (high expression level of TfR) and HepG-2 (low expression level) and one normal mouse cell line (3T3) with different levels of TfR as shown in Fig. 8. The fluorescent probe was obviously ingested only by Hela cells, and noticeable fluorescence was observed after 4 h of incubation.

Fig. 8
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Fabrication of the Tf-AuNCs/GO conjugation as a turn-on NIR fluorescent probe for bioimaging in cancer cells with TfR over expression [94]

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Sahoo et al. have developed a quick one-step green synthesis (2 min) of highly luminous AuNCs on DNA, using a single heating and cooling cycle as in polymerase chain reaction (PCR). The intensity of luminescence nanoclusters was found to increase with the number of DNA, offering an easy way to quantify DNA (Fig. 9). As a powerful fluorescent probe for DNA quantification, the capability of AuNCs is shown in two different cancer cell lines included HeLa and A549 [95]. The formation of AuNCs was found to be influenced by the amount of precursors (HAuCl4) used in synthesis. The intensity of luminescence emissions and the quantum results of nanoclusters are seen based on the cluster dimensions formed on various amounts of gold. The AuNCs were prepared by different base pairs, which consisted of A, T, G, and C, and produced the same luminescence for different base pair compositions and the same sequence lengths. Furthermore, the identification of the emission-intensity dependence of nanoclusters on DNA quantities provides a unique way of testing. Analysis of gene amplification and relative expression can be obtained. Moreover, the biocompatibility of AuNCs further emphasizes its use as a probe compared with the traditional cytotoxic properties of dyes. Quantitative analysis of the level of gene expression in various cancer cell lines can be used to demonstrate a simple, portable, and low cost equipment as an alternative to complicated, powerful, and expensive PCR energy machine. Furthermore, with the uses of luminescent AuNCs as signal-generating agents, this tool enables reverse transcriptase PCR and array-based analysis of multiple genes/proteins at the same time using switchable holders and specially designed software. Devices and approaches were developed to assess gene profiles associated with apoptosis in HeLa cancer cells and also to measure the expression of glutathione-S-transferase (GST) protein and GST-tagged human granulocyte macrophage colony-stimulating factor (GSThGMCSF) recombinant protein extracted form Escherichia coli [96].

Fig. 9
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The method for the synthesis of luminescent AuNCs by emulating the PCR condition [95]

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High-speed preparation of biocompatible signal-producing agents of AuNCs in DNA and proteins allows qualitative detection. Moreover, it provides synthetic methods of AuNCs as common probes for both DNA and protein studies (in fluids as well as samples on membranes), PCR amplicon analysis, and membrane-based researches in a single instrument. The instrument is capable of delivering 95% PCR amplification efficiency as compared to commercially available machines. Most important, the materials are all environmentally friendly. Taking the advantages, integrated tool and approach can create a novel application to existing techniques with the incorporation of nanotechnology and biology.

Nguyen et al. develop double ligand of stabilizing AuNCs and fabricate AuNCs/graphene nanocomplex as a “turn-on” fluorescent probe to detect matrix-related matrix metalloproteinase-9 cancer [97]. A smooth, one-step method was investigated for the biomedical application of AuNCs using peptides and mercapto undecanoic acid as co-templating ligands. The peptide with metalloproteinase-9 cleavage site serves as a stabilizer and also as a targeting ligand for enzyme sensing. With enzymes, because of the excellent quenching properties and negligible background of graphene oxide, the AuNCs/graphene nanomaterial produces a strong “turn-on” fluorescent response, which is highly correlated with enzyme concentrations. The limit of detection of the nanomaterial is 0.15 nM for enzyme. The fluorescent nanomaterial was successfully demonstrated for detection of “turn-on” metalloproteinase-9 secreted from MCF-7 cancer cell with high sensitivity and selectivity. Furthermore, the fluorescent AuNCs provide significant reductions in time, cost, and sensory complexity compared to previous studies. The platform has also shown great potential for detecting different biological molecules in diverse fields including environmental and analytical researches. Similarly, Song et al. develop the label-free, sensitive, and simple method for detecting protein kinases based on the selective aggregation of phosphorylated-gold nanoclusters peptides (AuNCs-peptides) induced by the coordination of Zr ion [98]. The AuNCs were prepared by peptides without a strong reducing agent, which prevents peptides from being disturbed. A study of label-free, green, sensitive, and simple fluorescence using the AuNC-peptides to measure the activity of the protein kinase CK2 has been developed. Compared with the recent established kinase fluorescence test, the uses of AuNC-peptides have several important advantages, including label-free, green, and simple experimental processes.

Selvaprakash et al. develop AuNCs using low-cost chicken egg white proteins (AuNCs@ew) as a switch-on sensing probe to detect phosphate-containing metabolites such as adenosine-50-triphosphate (ATP) and pyrophosphate (PPi) [99]. A cost-effective and straight-forward approach to producing fluorescent AuNC probes for phosphate-containing molecules such as ATP and PPi has been obtained. By adding cheap egg whites with tetrachloroauric, AuNCs@ew can be easily synthesized by microwave heating. In this work, AuNCs@ew mainly dominated by AuNCs@ovalbumin through careful characterization. Since ovalbumin is a glycoprotein and contains abundant glycine ligands, the possibility for the use of AuNCs@ew as the fluorescent probes for ConA, which contains the glycans binding site, has been successfully proven in Selvaprakash’s work.

Wu et al. use bovine serum albumin (BSA) and GSH to synthesize gold nanoclusters (BSA/GSH-AuNCs) with excitation and emissions at 330 nm and 650 nm, respectively [100]. In this approach, BSA and GSH serve primarily as a limitation and reducing agents, respectively. With the help of GSH, only 30 μM BSA is needed to synthesize photostable BSA/GSH-AuNCs. With the use of GSH, the use of large amounts of expensive proteins such as BSA and transferrin is no longer necessary for the development of fluorescent proteins/GSH-AuNCs. This strategy provides a low-cost approach for the synthesis of protein-AuNCs and also simplifies the refining of the established AuNCs. Wu et al. also found that quenching triggered by NO2− at pH 3.0 was efficient and specific. With high salt tolerant, sensitivity, and selectivity, BSA/GSH-AuNCs have great potential for measurement of complicated NO2 samples. Cao et al. investigate pH-induced fluorescence changes from AuNCs@BSA and appropriate conformational changes of ligand proteins by fluorescence, circular dichroism (CD), and IR spectral measurements. In this work, BSA in AuNCs@BSA undergoes identifiable conformational changes at the level of secondary and tertiary structures. CD and IR results interpret a significant change from the second structure on extreme acidity and alkaline, where more irregular structures are obtained [101]. The difference in secondary structural change trends between AuNCs@BSA and the original BSA was shown. The extreme alkaline condition (pH 11.43) induces a change from exposure to the buried helix. Moreover, the large tryptophan fluorescence gap between AuNCs@BSA and the original BSA implies that the gold nuclei live near tryptophan in the BSA. This study lays the groundwork for understanding the behavioral conformation of ligand proteins in conjugated AuNCs.

Ghosh et al. investigate the effects of AuNCs on CD and enzymatic activity of α-chymotrypsin (ChT) (against substrate hydrolysis, N-succinyl-l-phenylalanine p-nitroanilide) [102]. The CD spectrum shows that on binding to AuNCs, ChT is completely exposed, producing almost zero ellipticity. The ChT-coated AuNCs show virtually no enzymatic activity. The additional GSH or oxidized GSH restores enzyme activity a ChT by 30–45%. The activity of ChT is irreversibly lost on the binding surface of AuNCs. This lost activity can be recovered when ChT closes AuNCs treated with GSH or oxidized GSH. In the cell, the enzyme activity can be revived by GSH as shown in this work. Since cancer cells are characterized by elevated levels of glutathione, there will be differences in the absorption of enzyme-lined gold groups between cancer cells and normal cells.

The novel technique of fluorescence-guided surgery has entered the surgical process to help operators make the decision whether the tissues need to be resected or preserved during surgery [103]. These achievements could establish a paradigm shift in cancer surgery for great improvement of patient outcome. Recent research progress in this field has focused on the use of fluorescent AuNCs conjugated with diatrizoic acid and target-specific AS1411 aptamer as a fluorescence-guided probe for providing precise guiding during tumor tissue resection. In vivo experiments have demonstrated that the tumor location in CL1-5 tumor-bearing mouse has been observed from the clear CT image using the AuNC conjugates as a molecular imaging contrast agent. More importantly, the clearly visible orange-red fluorescence of AuNC conjugates has been utilized to help the resection of the CL1-5 tumor by intraoperative fluorescence guidance. The strong fluorescence enhancement of resected tumor is based on in vivo imaging system data to prove the successful molecular targeting using fluorescent AuNC conjugates. This work has demonstrated great advantages of using the target-specific fluorescent AuNC conjugates in vivo that are able to provide long-term fluorescent imaging times, high photostability, dual imaging functions, and feasible surface modifications with specific-targeted molecules in comparison to most organic contrast agents currently used. In addition, this work has brought an advanced concept in the field of biomedical imaging and therapeutics using functionalized AuNCs.

Conclusions

Overall, we have provided a mini review of the recent advances in fluorescent AuNCs prepared with small molecules, polymers, and biomacromolecules for the applications in bioimaging, detection, and therapy (Table 1). These works have shown that fluorescent AuNCs can be promising fluorescent probes due to their unique properties such as excellent biocompatibility, high photostablility, and easy surface modification. Although AuNCs have been demonstrated in various biomedical applications, however, their fluorescence quantum yields (QYs) are still low (usually less than 20%). The first challenge to extend the applications of AuNCs is focused on the preparation of AuNCs with high fluorescence QY. With low fluorescence QY, the synthesis of AuNCs with uniform size will be an alternative way to improve their fluorescence QY. Furthermore, with the uniform size, fluorescent AuNCs with a narrow emission spectrum will increase their benefit in biomedical applications. The second challenge for AuNCs is the control of ligand on their surface because the chemical and physical properties of AuNCs can be significantly affected by their surface modification. Therefore, the theoretical and practical studies of AuNCs are still needed to have a better understanding of their structure, optical characteristic, and physicochemical property. Especially, for physicochemical property, recent studies have proven that AuNCs are potential fluorescent probes for biosensing, bioimaging, and cancer therapy. Accordingly, to realize the biomedical applications, we still have a lot of works to push the biomedical applications of AuNCs in imaging, detection, and therapy. Overall, with the great efforts, we believe that AuNCs will be served as a significant fluorescent probe in biomedical application in the near future.

Table 1 Colors, detection targets, and applications of AuNCs with various ligands in this review
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