Recent Advances in Metal Nanoclusters-based Fluorescent Platforms for Biosensing

WANG Meng-ke,SU Xing-guang

(Department of Analytical Chemistry,College of Chemistry,Jilin University,Changchun 130012, China)

Abstract:Biosensing platform is one of the branches of chemical sensing platform,which is sensitive to the biomolecules and can convert the concentration changes of biomolecules into electric signal for the sensing purpose.Among the various biosensing platforms,fluorescent nano-biosensing platforms have attracted widely attention due to their advantages of high sensitivity,good selectivity,operational simplicity,low cost,real-time monitoring,etc.In recent years,with the rapid development of nanotechnology,nanostructured materials(nanomaterials) exhibit some special advantages in the field of biosensing.Compared with the traditional materials,nanomaterials exhibit unique physical and chemical properties,such as optical,electrical,mechanical,catalytic and magnetic properties.As a novel type of multifunctional nanomaterials in nanoscience and nanotechnology,metal(e.g.,Au,Ag,Cu,and their alloys) nanoclusters(MNCs) are typically composed of several to dozens of metal atoms whose core sizes are usually below 2 nm.Benefiting from their unique properties of strong photoluminescence,facile preparation and surface functionalization,excellent biocompatibility,ultra-small size and low toxicity,MNCs have been applied in many fields such as energy catalysis,medical treatment,electronic devices and biosensing.Especially,they have extremely outstanding fluorescent properties of large Stokes shift,tunable fluorescence emission,high optical stability and fluorescence quantum yield,compared with the traditional fluorescent organic dyes,proteins,quantum dots and other fluorescent materials.Based on the above properties,MNCs have been widely used as fluorescent nanoprobes in the field of biosensing.In this review,the latest development of MNCs based fluorescent nano-biosensing platforms on the basis of various construction mechanisms were presented.The applications of MNCs in the field of sensing ions,biomolecules,pH and temperature were introduced.It is believed that this review could provide some novel insights and theoretical guidance to construct more prospective biosensors based on different designed sensing mechanisms.

Key words:fluorescent nanoprobe; metal nanoclusters; biosensing; nanomaterial

In recent decades,due to their unique catalytic,optical,electrical,magnetic and chemical properties,metal nanoparticles(MNPs) with the size between 2 nm and 100 nm have achieved great achievements in various research areas of chemical sensing,catalysis,biology,medicine,and environmental sciences[1-2].However,when the size of the MNPs further reduces and approaches the Fermi wavelength of conduction electrons,the continuous energy bands of MNPs will break down into discrete energy levels due to the strong quantum confinement effect,which results in significantly different optical,electrical and chemical properties compared with the traditional materials.These MNPs with a size less than 2 nm are defined as metal nanoclusters(MNCs)[3-5].MNCs(e.g.,Au,Ag,Cu,and their alloys),as an important transition between large-sized metal nanoparticles and single metal atoms,are usually composed of several to dozens of metal atoms.Especially,due to the size comparable to the electron Fermi wavelength,MNCs exhibit electronic discrete energy levels and molecular-like properties[6-8],such as inherent magnetism,highest occupied molecular orbital and lowest unoccupied molecular orbital(HOMO-LUMO) conversion,quantized capacitance charging effect,high catalytic activity,large Stokes shift and strong luminescence properties.Benefiting from these unique and excellent properties,MNCs have widely used in various fields such as biosensing,medical treatment,electronic devices,and energy catalysis[9-12].

Characteristically,MNCs no longer exhibit obvious surface plasmon resonance absorption similar to the traditional materials,but show strong luminescence from the visible to the near-infrared region[13].With the development of an increasing number of efficient and convenient synthesis methods,such as etching technique,template-based synthesis,metal replacement,ligand exchange and so on[14-16],fluorescent properties,as one of the charming properties of MNCs,have received extensive attention from researchers.Compared with the traditional organic dyes,quantum dots and other fluorescent materials,the excellent properties of tunable fluorescence properties from the visible light region to the near-infrared region,high quantum yield,excellent light stability,simple preparation method and facile surface functionalization make MNCs act as a promising type of fluorescent nanomaterials,which have been widely used in the design and preparation of various types of fluorescent nanoprobes[17-19].

In recent years,some review articles have been dedicated to the applications of fluorescent MNCs,such as the review on the fluorescent properties and representative applications of Cu nanoclusters for sensing and bioimaging[20],recent advances in the templated synthesis of MNCs and their fluorescent applications[21],the mini review on the luminescence mechanism and synthesis of fluorescent Au nanoclusters and their applications in bioimaging and biosensing[22],and so on[23-25].Apparently,these reviews are mainly focused on the synthesis and properties of MNCs with brief discussions on the application of fluorescent MNCs,and the review on construction mechanism for sensing platform was rarely reported.Herein,compared with the previously reported reviews,this review will pay more attention on the different construction mechanisms for the MNCs-based biosensing systems for the purpose of detecting heavy ions,trace elements,drugs,biomolecules,pH and temperature.

1 Application of MNCs in biosensing

1.1 Ion detection

In recent years,MNCs based fluorescent sensing platforms have great prospects in the applications of convenient,accurate and specific analysis of ions.For example,Kang's research group[29]designed a ratio fluorescence sensing platform for Pb2+detection in porphyra based on CuNCs/nitrogen-doped carbon quantum dots(CuNCs/CNQDs) nanoprobes.In this nanoprobe,the fluorescence of CuNCs was enhanced by Pb2+owing to the aggregation-induced emission enhancement(AIEE) between Pb2+and CuNCs.The fluorescence of CNQDs remained almost unchanged in the coexistence with Pb2+,providing the self-calibration signal.According to the change of fluorescence intensity ratio,the sensitive detection of Pb2+was achieved with a low limit of detection(LOD) of 15 nmol/L by employing the CuNCs/CNQDs nanoprobe.Based on the ion-mediated AIEE of MNCs,Fu's team[30]developed a one-step sensing method of S2-with a specific response.Cu2+exhibited the ability to combine with 3-mercaptopropionic acid-stabilized CuNCs(MPA-CuNCs) forming aggregated Cu2+@MPA-CuNCs,which resulted in the significant fluorescence enhancement of MPA-CuNCs.The strong affinity of S2-to Cu2+restrained the aggregated Cu2+@MPA-CuNCs,causing the fluorescence quenching of the system.Based on the above design,the sensitive detection of S2-was achieved with a LOD of 26.3 nmol/L.Additionally,the analysis of S2-in food additives was successfully performed by this assay.

Benefiting from the convenient and rapid synthetic route of MNCs,some promising sensing systems realize the detection of ions based on the in-situ formation of MNCs.For example,Nie et al.[33]constructed a fast,simple,and label-free fluorescent sensor for the determination of SCN-based on the in-situ formation of DNA-templated CuNCs(DNA-CuNCs).As shown in Fig.1B,in the absence of SCN-,stable fluorescent DNA-CuNCs were formed via the reduction of Cu2+by ascorbic acid and DNA acting as the template.However,SCN-reacted with Cu2+to result in Cu(SCN)2precipitation.Therefore,the consumption of Cu2+inhibited the generated DNA-CuNCs and further weakened the fluorescence of the system.This method can directly determine SCN-without the requirement of the pre-synthesized fluorescent probes.The LOD of SCN-was 4.94 μmol/L.

1.2 Drug analysis

Drug is the special substance for human disease prevention,treatment and rehabilitation,accordingly,the content or quality of drug is closely related to therapeutic effect[37].In order to take the appropriate dosage of drug and determine the effective content of commercial drug,accurate and sensitive quantitative detection of drug is crucial for life security.In addition,nowadays,antibiotic has been widely used due to their highly effective sterilization and anti-inflammatory functions.However,residual antibiotic will cause the pollution of surface water after a series of transfers,and the residual antibiotic in water or food will further pass through biological ingestion,which undoubtedly increases the resistance of bacteria to antibiotics in organisms[38].Therefore,the development of efficient antibiotic detection strategies is highly vital to protect human health and the ecosystem.

Borse et al.[39]successfully prepared PtNCs(CEW-PtNCs) via one-step approach with chicken egg white as the template and stabilizing agent.Besides,based on the aggregation of CEW-PtNCs induced by neuro drug carbidopa,a convenient “turn-off” fluorescence platform was constructed for the determination of carbidopa with a LOD of 1.71 μmol/L(Fig.2A).The analysis of carbidopa in pharmaceutical samples and biofluids was conducted with satisfactory performance.Zhu et al.[40]employed DNA-AgNCs as novel fluorescent probes to specifically recognize penicillamine.Penicillamine,containing a thiol,could combine with Ag in DNA-AgNCs to form the non-fluorescent ground state complex,which led to the aggregation and fluorescence quenching of DNA-AgNCs.The LOD of penicillamine was 8 nmol/L.Based on the quenching mechanism of IFE,Li et al.[41]designed a fluorescence “turn-off” strategy for the ultra-sensitive detection of antibiotic rifampicin with a low LOD of 16 pmol/L by utilizing glutathione(GSH) stabilized CuNCs as fluorescent probe.Besides,by integrating fluorescence quencher manganese dioxide(MnO2) nanosheets,Zhao et al.[42]prepared poly(methacrylic acid)-coated AgNCs(PMMA-AgNCs) and constructed a PMMA-AgNCs/MnO2nanosheets based fluorescent sensor for trace detection of tuberculostatic drug isoniazid(INH).As shown in Fig.2B,INH can reduce MnO2nanosheets to Mn2+,thereby inhibiting the fluorescence quenching effect of MnO2nanosheets on PMMA-AgNCs.The LOD of this method for INH was 476 nmol/L.This detection strategy was also used for the determination of isoniazid in human body fluids and pharmaceutical products.

Fig.2 Scheme of the one-step synthesis of CEW-PtNCs and the sensing application for carbidopa assay[39](A), and schematic diagram of the signal-on strategy for detecting isoniazid by using AgNCs/MnO2 nanosheets system[42](B)

In addition to the above single fluorescent signal based sensing modes,the ratio fluorescence sensing strategies were also designed and used for drug analysis.Our group[43]constructed a dual-emission ratio probe for bleomycin(BLM) analysis based on nitrogen doped graphene quantum dots@AuNCs assembly(NGQDs@AuNCs).As revealed in Fig.3A,fluorescence intensity of AuNCs can be quenched by Cu2+due to the Cu2+-triggered aggregation of AuNCs.However,fluorescence intensity of AuNCs restored in the presence of BLM because of the strong affinity between Cu2+and BLM.Meanwhile,fluorescence intensity of NGQDs remained unchanged throughout the process.Based on the change of fluorescence intensity,BLM can be sensitively detected with a low LOD of 0.27 nmol/L.Furthermore,this assay in the application of human serum samples was conducted.Based on the peptide conjugated with aggregation-induced emission luminogens(TPE-p) and vancomycin(Van) aptamer-modified AuNCs(AuNCs-apt),Shi et al.[44]established a novel ratio fluorescence biosensing platform for the detection of glycopeptide antibiotics Van(Fig.3B).The specific recognition and high-affinity binding of Van to peptide(p) and aptamer(apt) promoted the formation of aggregated TPE-p/AuNCs-apt/Van complexes,which resulted in the significant fluorescence enhance of TPE-p and slow fluorescence increase of AuNCs-apt.With the continuous increase of Van,the fluorescent color of TPE-p/AuNCs-apt/Van system changed from red to orange and blue.The LOD for Van was 1.88 nmol/L.The determination of Van in human serum and microdialysate samples was successfully performed by this sensing system.

Fig.3 Schematic illustration for bleomycin analysis[43](A),and schematic diagram of the sensors for vancomycin detection[44](B)

1.3 Biomolecule sensing

Biomolecules generally refer to the various specific molecules of organisms.Divided by the molecular weight,biomolecules mainly include small biological molecules,such as lipids,vitamins,amino acids,catecholamines,peptides,etc.,and large biological molecules,such as proteins,polysaccharides,nucleic acids and bio-enzymes.Biomolecules participate in various life activities and play an indispensable role in maintaining the normal operation of numerous physiological functions in the human body[45].Importantly,most of biomolecules are regarded as biomarkers for the early disease diagnosis[46].Therefore,accurate identification and quantitative analysis of biomolecules are of great significance in related biomedical fields.Due to the excellent physicochemical properties of MNCs,establishing MNCs based fluorescent sensing platforms from different design perspectives for detecting target biomolecules has aroused more and more research interests.

Based on the coordination,oxidation,reduction or other chemical binding effects between the biomolecule and the central metal of the MNCs trigger the fluorescence changes of the MNCs,the researchers have designed versatile sensing strategies for detecting various target biomolecules.Our group[47]successfully synthesized papain-protected bimetallic gold/silver nanoclusters(Au/AgNCs) and applied Au/AgNCs to design a “off-on-off” fluorescence sensing platform for detecting ascorbate oxidase(AAO) based on the oxidant H2O2-triggered fluorescence quenching of Au/AgNCs.The addition of ascorbic acid(AA) with reducibility can effectively inhibit the fluorescence quenching of Au/AgNCs.Furthermore,AAO could catalyze the oxidation of AA,resulting in the fluorescence decrease of Au/AgNCs.The AAO activity can be conveniently monitored by measuring the fluorescence intensity change of Au/AgNCs with a LOD of 1.72 mU/mL.Huang's research group[48]established a fluorescence “turn-off” sensor platform to realize the determination of glucose oxidase based on the oxidation of Cu at the center of CuNCs/ZIF-8 nanocomposites by the H2O2that was produced by the glucose oxidase-modulated catalytic oxidation of glucose,which cause the fluorescence quenching of CuNCs.The LOD of glucose oxidase was 0.035 U/L.Similarly,based on the cholesterol oxidase-catalyzed oxidation of cholesterol to generate H2O2,Baker et al.[49]employed protein-templated AuNCs as probes for the detection of cholesterol with a LOD of 12 μmol/L.

By integrating the reducibility of sulfhydryl group to reduce the Au of AuNCs and the catalytic hydrolysis of acetylthiocholine to produce thiocholine by acetylcholinesterase(AChE),Yu's group[50]designed a novel and convenient photonic crystal/BSA-AuNCs/fluorescein-based ratio fluorescence sensing platform for detecting acetylcholinesterase(AChE) with a LOD of 0.027 mU/mL.On the basis of the AIEE triggered by thiol biomolecules through Au-S coordination,our group designed a fluorescence detection platform for cysteine detection[51].The Ce3+bound with the carboxy groups of GSH-capped AuNCs,which resulted in a significant fluorescence enhancement of AuNCs due to the AIEE effect.Cysteine(Cys),performing as the bridge,interacted with Ce3+by carboxyl group and linked with AuNCs by Au-S bond.Through the linkage of cysteine,the aggregation degree of AuNCs was more intense owing to the smaller spatial structure of cysteine than that of GSH,resulting in the increase of AIEE effect with the great enhancement of fluorescence intensity.Cysteine can be quantified via this effect with a LOD of 0.15 μmol/L.

Some biological molecules,such as nucleic acids(DNA or RNA),polysaccharides and polypeptides,can perform as the templates for synthesizing MNCs[52],as a result,by skillfully applying the hybridization between nucleic acids,the highly specific binding of nucleic acid aptamers to targets,the specific catalysis of biological enzymes on nucleic acids,researchers have elaborately designed many MNCs based sensing systems for specific biomolecules.Li's team[53]applied DNA hairpin-templated AgNCs to construct a visual and universal fluorescence platform for detecting target DNA based on exonuclease(Exo) Ⅲ assisted recycling amplification(Fig.4A).In this design,an unlabeled DNA hairpin was designed as a molecular beacon(MB),and the complete DNA hairpin can be used as a template to synthesize AgNCs with high fluorescence intensity.When MB recognized and bound to the target DNA,forming a three-way junction structure,the hairpin structure will be recognized and cleaved by Exo Ⅲ,releasing the target DNA for cyclic cleavage.Due to the broken hairpin structure can not support to form AgNCs,the fluorescent probe was in the “turn-off” mode,which achieved the sensitive detection of the target DNA with a LOD of 2.8 nmol/L.Our group[54]used DNA-templated CuNCs as the simple and sensitive fluorescence probe to detect protein kinase A(PKA)(Fig.4B).This assay was based on the strong interaction between adenosine triphosphate(ATP) aptamer and ATP.In the presence of ATP,the formation of CuNCs was blocked due to the lack of effective substrate,and resulting in the fluorescence decrease of CuNCs.However,with the addition of PKA,the fluorescence of CuNCs recovered because of the transformation of ATP to adenosine diphosphate(ADP) by PKA and the incapable combination of ADP with ATP aptamer.The PKA can be effectively monitored according to the variation of fluorescence signal with a LOD of 0.041 U/mL.Besides,based on probe bonding with the target chain,Qian et al.[55]constructed a label-free DNA stabilized AgNCs probes with the identification and signal output capabilities for specifically detecting target Norovirus RNA with a LOD of 18 nmol/L.Zhao's research group[56]designed a platform for simple detection of prostate-specific antigen(PSA) based on that the specific recognition of PSA with aptamer DNA blocked the in situ formation of DNA-AgNCs.The LOD of PSA was 1.14 ng/mL.

Due to that some biological molecules for synthetizing MNCs are the specific substrates of bio-enzymes,based on the degradation of biological molecules by bio-enzymes to block the generation of MNCs,specific detection of target bio-enzymes can be achieved.Jia's group[57]synthesized CuNCs(CS-GSH-CuNCs) by using polysaccharide chitosan(CS) as confining agent and GSH as reducing-cum-protecting ligand.Then,the CS-GSH-CuNCs were applied for the determination of lysozyme(Lys) .Due to the electrostatic confinement effect of positively charged CS,the fluorescence of CS-GSH-CuNCs was much higher than that of GSH-CuNCs prepared without chitosan.The specific hydrolysis of chitosan by Lys significantly inhibited the fluorescence enhancement effect of CS on GSH-CuNCs.Based on the fluorescent quenching caused by Lys,Lys can be sensitively detected with a low LOD of 1.6 nmol/L.In addition,our group[58]reported a detection platform for Exo Ⅲ based on Exo Ⅲ hydrolyzed DNA to block the generation of DNA-templated CuNCs with a LOD of 0.02 U/mL.Based on the fact that the phosphorylation of the peptide substrate by protein kinase A prevented the peptide template of AuNCs from being hydrolyzed by carboxypeptidase,Qiu's team[59]used peptide-stabilized AuNCs as fluorescent probes to detect protein kinase A with a LOD of 0.004 U/mL.

With the assistance of fluorescence quenchers,such as nanoparticles,nanosheets,small organic molecules,ions,etc.,a series of of fluorescence sensing platforms were constructed for specifically and sensitively determining of target biomolecules.Based on the fluorescence resonance energy transfer(FRET) between polydopamine nanoparticles(PDANSs) and dual-color AuNCs(P1 and P2),Luo et al.[60]designed a novel sensing platform that can simultaneously quantitatively detected two miRNAs(miRNA-21 and let-7a) with deoxyribonuclease I(DNase I)-assisted target recycling signal amplification(Fig.5A).PDANSs possessed the efficient ability to adsorb P1 and P2,resulting the effective fluorescence quenching of P1 and P2.After adding the complementary target miRNAs,probes P1 and P2 hybridized with target miRNAs and detached from the surface of PDANSs.Then P1 and P2 were cleaved by DNase I,releasing target miRNAs to combine another pair of probes and repeat the cyclic cleavage reaction,leading to a prominent fluorescence enhancement.Through the recovery of the fluorescence signal of the system,miRNA-21 and let-7a were simultaneously detected with the LODs of 4.2 pmol/L and 3.7 pmol/L,respectively.This method was successfully used for miRNA-21 and let-7a detection in the serum of healthy humans and breast cancer patients.Our group[61]developed and applied chondroitin sulfate-templated Au/AgNCs to establish an ingenious sensing platform for sensitive analysis of hyaluronidase(HAase) with a LOD of 0.3 U/mL under the attendance of gold nanoparticles(AuNPs).As revealed in Fig.5B,AuNPs can distinctly quench the fluorescence of Au/AgNCs due to IFE.With the introduction of protamine(PRO),the positively charged PRO can bind to negatively charged AuNPs via electrostatic attraction,which led to the aggregation of AuNPs and the fluorescence recovery of Au/AgNCs.After the preferential interaction of PRO with negatively charged hyaluronic acid(HA),AuNPs disaggregated and quenched the fluorescence again.However,the hydrolysis of HA by HAase prevented HA from interacting with PRO,resulting in the aggregation of AuNPs and weaken the IFE of AuNPs on Au/AgNCs.This “turn-on” sensing system can also be utilized to detect HAase level in human serum samples.

Fig.4 Illustration for enzymatic amplification DNA detection[53](A),and schematic illustration of the strategy for the detection of PKA activity based on the fluorescent CuNCs[54](B)

Based on the reducing substance ascorbic acid produced in alkaline phosphatase-medicated hydrolytic system to inhibit the fluorescence quenching effect of cobalt oxyhydroxide(CoOOH) nanosheets on CuNCs,Liu's research group[62]constructed a simple fluorescent method for the detection of alkaline phosphatase with a LOD of 0.1 mU/mL by integrating CuNCs and CoOOH nanosheets(Fig.5C).Liu et al.[63]proposed a simple fluorescent assay for the sensitive detection of GSH with a LOD of 2.2 μmol/L based on the reduction of MnO2nanosheets by GSH to inhibit the MnO2nanosheets-triggered fluorescence quenching of AuNCs.Based on the fluorescence quenching of Au/AgNCs caused by the IFE between 2,3-diaminophenazine(DAP) and Au/AgNCs,Su's research group[64]specially designed a ratio fluorescence platform to detect metabolism uric acid in human blood(Fig.5D).Urate oxidase catalyzed the oxidation of uric acid to produce H2O2.In the presence of H2O2,horseradish peroxidase catalyzeso-phenylenediamine to produce fluorescent DAP,and DAP quenched the fluorescence of Au-AgNCs through IFE.Based on the change of the fluorescence ratio of the system,uric acid was detected with a LOD of 5.1 μmol/L.Based on the fluorescence and mimicking peroxidase properties of AuNCs and the glucose/glucose oxidase system to produce H2O2,Chu's research group[65]applied AuNCs to catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine(TMB) in the presence of H2O2.The produced oxidation product oxTMB was the effective fluorescence quencher of AuNCs.According to the fluorescence decrease of AuNCs,glucose was detected with a LOD as low as 0.01 nmol/L.Based on the fact that the binding of phosphate-containing biomolecules to Cu2+inhibited the Cu2+-triggered fluorescence quenching of AuNCs,Selvaprakash's group[66]proposed a convenient fluorescence strategy for the determination of adenosine triphosphate with a LOD of 19 μmol/L.

1.4 Temperature and pH sensing

Temperature controls the numerous kinetic reactions of intracellular biomolecules,which is the widely used as a fundamental thermodynamic parameter to monitor the reactive states of the biochemical reactions in living cells[67-68].The variations of the intracellular temperature pervade the profound understanding of the most basic of physical,biological and chemical phenomena.Accordingly,the precise measurement of intracellular temperature is conducive to diagnose a variety of diseases at the cellular level and develop new types of methods of the disease treatment.On the other hand,research interests on pH sensing platforms have greatly increased because intracellular pH plays the pivotal role in regulating many biological processes,such as maintaining the biological functions of proteins and performance of intracellular organelles.The abnormality of pH is one of the cause of dysfunctions of organelles,which leads to the cardiopulmonary and nervous system diseases[69].The pH value has been widely used as an indicator of many diseases in medical diagnosis.Therefore,it is of considerable significance to develop new noninvasive and accurate diagnostic techniques to measure pH with high spatiotemporal resolution.In recent years,MNCs also have broad application prospects in the field of temperature and pH sensing.

Zhou et al.[70]proposed an attractive type of dual-fluorescence DNA-templated AgNCs pair(A-NCP and B-NCP) for sensitive temperature sensing(Fig.6A).With the temperature rising,the A-NCP fluorescent color changed from orange to yellow,and the B-NCP fluorescent color varied from orange to colorless.As they assembled into AgNCs pairs,the two single-stranded segmental AgNCs were integrated together,which result in the dramatic variation of fluorescence properties.The temperature increase-triggered dehybridization induced the separation of two pieces of segmental AgNCs,which was the account for the temperature-sensitive phenomenon.The AgNCs pair was also used to monitor the temperature in human breast cancer cells.Based on the formation of reversible aggregates at high pH values caused fluorescence quenching of trypsin-stabilized CuNCs,Huang's group[71]reported a sensitive,reversible and fast sensor for reversible pH-sensing with a wide pH range of 2.02-12.14(Fig.6B).

Fig.6 Scheme of temperature response by designing AgNCs pairs[70](A) and schematic illustration of the preparation and application of trypsin-templated CuNCs for the pH sensing[71](B)

Zhang's group[72]developed the captopril-stabilized AgNCs as fluorescent probes for reversible pH and temperature sensing.The increase of temperature induced the enhancement of nonradiative decay and the pH triggered protonation and deprotonation of carboxyl groups on the AgNCs,which were the main factors for the fluorescence quenching of AgNCs.This platform can achieve the sensitive measurement of temperature in the range of 10-45 ℃ and the pH range of 2.08-6.06.In addition,Wang et al.[73]proposed a simple and rapid method to synthesize bimetallic Au/Ag-NCs and applied them to design a sensing platform that simultaneously responded to pH and temperature.In the range of 15-50 ℃,the fluorescence intensity of Ag/AuNCs decreased with increasing temperature and possessed a reversible response to the temperature.The temperature resolution of Au/AgNCs was as high as 0.1-0.5 ℃.In addition,based on the good biochemical stability and fluorescence stability of Ag/AuNCs,they constructed a ratio fluorescence sensor for pH detection by applying fluorescein thiocyanate(FITC) as the measurement probe and Ag/AuNCs as the internal standard reference probe with a linear pH range of 6.0-8.5,and the sensor system was applied for pH sensing detection in HeLa cells.

2 Conclusions and future prospects

In this review,we have systematically summarized recent research progress on applications of MNCs in biosensing.The unique characteristics of ultrasmall size,strong fluorescence,large Stokes shift,excellent stability,good biocompatibility and tunable fluorescence emissions create a wide range of opportunities for fluorescent MNCs to be exploited in biosensing application.In spite of the considerable progresses,a variety of challenges remain and need to be explored.Firstly,some sensing systems are based on the ion-caused quenching or enhancement of fluorescence,which might be influenced in the presence of other ions or ionic polymers.As a result,the anti-interference capability of the methods requires to be systematically investigated.And more sensors with superior specificity should be developed.Secondly,most of the applications of the sensing strategies are still on the laboratory research level;thus,the following exploration direction is to exploit new avenues for designing next-generation smart sensors for the widespread clinical application,especially for the point-of-care test.Thirdly,with the increasing health concerns,it is also urgent to develop portable detection equipment based on the excellent properties of MNCs,such as home test kits,to realize the early prevention and diagnosis of diseases.