Recent applications of fluorescent nanodiamonds containing nitrogen-vacancy centers in biosensing

Abstract Fluorescent nanodiamonds (FNDs) with nitrogen-vacancy (NV) centers have been extensively studied in numerous fields because of their distinct magneto-optical properties. The NV center is a perfect candidate for a nanosensor because of its stable photoluminescence and manipulable spin state by microwave/magnetic field. Considering the controllable sizes (5–100 nm), abundant surface groups, and good biocompatibility, FNDs are valuable in biosensing to study the physiological activity at the cellular scale. This review summarizes the recent applications of FNDs in detecting physiological parameters (such as temperature, pH) as well as proteins, free radicals, viruses, etc. Highlights include the development of FND-based biosensors and the NV center transduction system that responds to signal changes or concentrations fluctuations of target species.


Introduction
Carbon is not only the foundational component to life, but also the primary component of coal, oil, and natural gas. Especially, carbon nanomaterial is a highly active research realm due to its remarkable optical, electrical, mechanical, and thermal capacities [1]. On the basis of their dimensionality, carbon nanomaterials include zero dimensional (fullerenes, carbon quantum dots), one-dimensional (carbon nanotubes, nanowires), two-dimensional (single-layer graphite), and three-dimensional materials [2,3]. Considering the different hybridizations of carbon atoms, there are also a variety of graphyne, graphene, diamonds and so on [4][5][6]. Among these carbon allotropes, nanodiamond (ND) is an important member and has important applications in many fields [7][8][9]. In recent years, fluorescent nanodiamonds (FNDs) containing certain point defects have attracted researchers' attention because of its unique optical and magnetic characteristics [10]. One of the most noteworthy defects is nitrogen vacancy (NV) defects center, in which a carbon atom in the ND lattice is replaced by a nitrogen atom, and a vacancy is formed around it [11]. Due to the special energy level structure, the FNDs can be excited by a light source with a specific wavelength and emit stable fluorescence [12]. Because the existence of extra magnetic field can make NV center's ground energy level splitting, the optical properties can couple with external magnetic field [13]. In addition, ND is considered to have good biocompatibility and surface functionalizability [14,15]. Inspired by these characteristics, researchers have developed FNDs-based optical probes, nanoscale sensors, and other sensing devices in the biological field [16]. This review focuses on the latest progress in the application of FNDs in the field of biosensors, and introduces the application of FNDs in temperature monitoring, pH detection, virus detection, and identification and quantitative detection of some specific physiologically active substances in biological scenes.

Nanodiamonds
ND has existed in nature for billions of years in meteorites, interstellar dust, planetary nebulae, crude oil, and various sedimentary layers of the earth. In the 1960s, the Soviet Union produced ND particles by explosive method for the first time. Since the late 1990s, scientists from all around the globe have looked into the unique structures and properties of NDs [17]. The primary particle size of ND can range from 1 to 150 nm, and its shape and structure might vary according on the preparation methods. The most widely applied preparation methods are detonation method and high temperature high pressure method (HPHT) [18]. Except for those two methods, NDs can also be prepared by chemical vapor deposition (CVD), microplasma-assisted synthesis, laser ablation, carbide chlorination, ultrasonic cavitation method, and plasma irradiation [17]. The raw ND usually has a sp 3 "diamond" inner core, covered by a sp 2 carbon shell [19]. Inheriting the superior characteristics of bulk diamond and unique properties of nanoparticles, NDs have superior hardness and Young's modulus, high thermal conductivity and resistivity, and good chemical stability, in combination with large surface area and high surface reactivity [20,21]. Very recently, ND has been proved to have good biocompatibility as well as low biological toxicity, sparking intense interest in drug loading and transportation [22,23].

Nitrogen-vacancy centers in nanodiamonds
Apart from those properties, FND exhibits unique optical and magnetic characteristics originating from the point defect [24,25]. The nitrogen-related NV center has drawn the greatest attention due to its red/NIR fluorescence for bioimaging and the magneto-optical properties [26]. Figure 1(A) shows the structure of NV center, in which a carbon atom in the lattice of nanodiamond is replaced by a nitrogen atom, forming a vacancy around it.
Although there are still neutral NV 0 and uncommon positively charged NV + , the research of negatively charges NVis more extensive [28]. Six electrons are involved in the NV center: two are provided by nitrogen atoms, and the other three are from dangling bonds of three carbon atoms around the vacancy. The sixth electron is usually captured by the nitrogen donor in the lattice, making the whole charge state appear as negative NV -. The energy level structure of NVis shown in Figure 1(B). It has a ground-state triplet ( 3 A), an excited-state triplet ( 3 E) and two intermediate-state singlets ( 1 A and 1 E). Both 3 A and 3 E contain the spin states of m s = 0 and m s = ±1 in its magnetic sublevels. The energy difference between the m s = 0 and m s = ±1 of 3 A states correspond to the microwave regime (2.87 GHz). The electron spin will leap to m s = ±1 from 0 sublevels when a resonant microwave radiation is applied. The two m s = ±1 sublevels are degenerate in a zero magnetic field, and split in the presence of external magnetic field via the Zeeman effect.
In terms of optical properties, the NVcenters emit bright red light ( 3 E→ 3 A) under excitation of green light with a wavelength of 532 nm, and exhibit the zero-phonon line (ZPL) at 637 nm which shows in Figure 1(C). The excited electrons return to the ground state via two possible pathways: (I) by the emission of 637 nm in the ZPL and phonon sideband; (II) firstly into the metastable singlet states ( 1 A) via intersystem crossing (ISC) and an infrared emission of a 1042 nm ( 1 E→ 1 A), further into the ground state with m s = 0 through ISC. The latter pathway results in a decrease in the emitted fluorescence intensity. It should be noted that the ISC rate from the m s = ±1 sublevel of the excited state to the singlet state is higher than that from the m s = 0 sublevel. Thus, the optical pumping can lead to a strong spin polarization into m s = 0 ground state after a few excitation-emission cycles. Besides, pumping with a resonant microwave frequency at 2.87 GHz promotes the electrons to the m s = ±1 states from m s = 0, which reduces the fluorescence intensity by about 30% after several cycles of excitation. That is to say, the spin states of NV − centers can be manipulated by microwave radiations and readout optically. Herein, optically detected magnetic resonance (ODMR) is generally used to detect the spin states and transitions between spin sublevels of NV − centers [29], as shown in Figure 2. The ODMR of NV − in diamond is unique and powerful, which has applications in magnetometry and sensing, biomedical imaging, and quantum information.  [25]. copyright © 2016 WilEY-VcH Verlag GmbH & co. KGaa, Weinheim (B) Schematic diagram of energy level structure of nVcenter [12]. copyright © 2014 ioP Publishing ltd. (c) Photoluminescence spectrum of a single nV − center [27]. copyright © 2017 Elsevier inc. all rights reserved.

Production of FNDs
To produce FNDs with NV − , an important issue is to create an appropriate number of internal defects in the diamond matrix. Commercial FND with NV defects are usually generated by irradiating HPHT diamond with high-energy proton (3 MeV) or electron (∼2 MeV) beams followed by thermal annealing at ∼800 °C. HPHT diamonds contain ≈100 ppm of single substitutional nitrogen impurity during growth. Lattice vacancies are created by high-energy particle irradiation which knocking carbon atoms out of their bonding positions. The vacancies can move under annealing (>600 °C), then captured by substitutional nitrogen, producing the NV centers [32,33]. However, this method requires highly complex and expensive equipment, which hinders the preparation of FNDs in the laboratory. Chang et al. [34] proposed a practical method to expand the production of FND by using a self-made prototype device composed of high flux and medium energy He + beams. Compared with the method used before, this device had increased the FND output by nearly two orders of magnitude, and could be safely installed and operated in ordinary laboratories. It should be noted that the annealing and irradiation process inevitably causes graphitization on the diamond surface, leading to quenching of the FND fluorescence. Before fluorescence the freshly prepared FNDs should undergo oxidation in air (around 450 °C) to remove surface graphite, followed by acid wash to remove metal and other impurities.
The nitrogen content and distribution in these particles are not optimized, and the nitrogen atoms in the nano-sized FND particles produced by these HPHT diamond particles are unevenly distributed. The uneven nitrogen distribution among the comminuted particles leads to uneven photoluminescence. One option to overcome this challenge is to use a bottom-up approach to synthesize FND with controlled nitrogen (and other dopants) content, including the method of synthesizing FND from non-traditional carbon precursors at high temperature and high pressure [32]. CVD is another technology for "bottom-up" synthesis of fluorescent nanodiamonds [35]. FND synthesized by this method does not require additional purification. The nanocrystals have a preselected density and position of light-emitting centers on the substrate, which is helpful to study the optical and spin characteristics of light-emitting centers in a single crystal. However, the low yield makes it unsuitable for mass production.

Temperature
Many physiological processes such as circadian rhythm regulation, cell metabolism, growth and development are closely related to temperature [36][37][38]. A current research hotspot is the use of the thermal effects of some materials in disease therapy, which entails raising the local tissue's temperature to kill cells at the location of the lesion [39][40][41]. Therefore, it is of great significance to construct a sub-micro-or a nano-scale thermometer to measure the temperature of local tissues, single cells and even sub-cellular scales in organisms when exploring the internal mechanism of life activities and developing new techniques of disease treatment.
Temperature significantly affects the spin resonance and coherence time of the NV center, which can be read out by ODMR technology [42]. This makes FND containing NV centers as an effective instrument for measuring temperature at the nanoscale with extreme sensitivity. By using ODMR within FNDs, Simpson et al. [43] successfully achieved mice intraneuronal enhanced signal-to-noise imaging and temperature mapping in 2017. The accuracy of temperature recording was improved to less than 1 K. The integration time was only a few tens of seconds, which allowed for quick  [30]. Rights managed by aiP Publishing. (B) a result of optically detected magnetic resonance spectra for a single nitrogen-vacancy defect at increasing magnetic field (from bottom to top) [31]. copyright © 2008, macmillan Publishers limited. all rights reserved. temperature detection of living biological specimens. Based on FND thermometer, Yukawa [44] preliminarily explained the relationship between temperature and stem cell differentiation in 2020 by combining confocal microscope with ODMR equipment. Thanks to the fluorescence characteristics of NV center, the appliance could enable cellular imaging and track the morphological changes of living cells in real time when measuring the temperature of stem cells. Fujiwara et al. [45] developed a FND thermometry system to monitor the temperature fluctuation of mobile NDs inside live worms of C. elegans. They added a correction filter to the ODMR system to address the positioning issues with FND particles inside cells and the measurement artifacts brought on by complicated multi cell architecture. In 2021, by integrating optical tweezers technology, Wu et al. [46] succeed in aggregating and fixing NDs in cells. They further realized the control of its concentration distribution and spatial position while solving the problem of NV thermometer movement.
Recent researches also focus on developing multifunctional thermometers by combining FNDs with heterogeneous materials, which can achieve heating and temperature detection simultaneously. Singo et al. [47] encapsulated FNDs with polydopamines (PDAs), with high photothermal conversion efficiency, to make a nanoheater/nanothermometer hybrid. Figure 3(A) is a diagram of this multifunctional device. Due to the PDA encapsulation, the temperature change was 4 times larger than pure FNDs under light excitation. By using these nanosensors, the intracellular thermal conductivities (κ cell ) of HeLa and MCF-7 cells were measured with a spatial resolution of about 200 nm. The thermal conductivity measure protocol is shown in Figure 3(B). For thermal conductivity κ, the parameters needed were calculated through the statistics collected from ODMR detection of PDA-FDA. As a result, the intracellular thermal conductivity of a single living cell (κ cell ) was successfully measured, which was close to κ oil , one sixth of κ water . Their experimental results provided important information for theoretical consideration of the role of heat transfer in cells. The FND-based heater-thermometer could be an effective tool to understand the control and regulation of heat in organisms at the single-cell level.
Wu et al. [48] prepared FND-based nanothermometer loaded with the photothermic anionic indocyanine green (ICG) to probe local temperature changes in living cells during photothermal therapy (Figure 4(A)). Compared to Sotoma's protocol, the heat generator ICG in this system served as photothermal agent to induced programmed cell death upon irradiation (Figure 4(C)). Unlike other luminescence-based thermometers (such as lanthanide-doped nanoparticles and quantum dots), the ICG-coated FND system measured temperature change by the shift of the zero-field splitting parameter via ODMR spectroscopy. Thus, this nanoscale sensor was less affected by environmental parameters such as pH, ionic strength, and intracellular viscosity. The temperature change curve measured by this method is shown in Figure 4(B). Therefore, FNDs could act as robust nanoscale thermometers with high sensitivity in biological systems, providing new insights into hyperthermia treatment at the nanoscale in intracellular environments.
Although NV center has been employed as a temperature measurement probe in living cells, there are still some issues that deserve attention. Microwave is necessarily used when recording ODMR spectra, which can interact with the intrinsic fields in diamond and exert a heating effect [49]. The excitation laser of 532 nm for the NV centers may also cause a heating effect resulted from the optical absorption of the biological body. The microwave and optical heating may impact the local heat-generation rate and temperature in biological samples. Additionally, a recent trend in FND-based temperature sensing is to develop multifunctional nano-thermometers by conjugating FND with other biological reagents [50]. Although much effort has been putting into developing advanced ODMR equipment and algorithms, a major challenge is the improvement of the temperature sensor's accuracy considering the staggering complexity of the physiological environment.

The pH value
The pH is a significant environmental parameter inside living body [51][52][53][54]. For instance, when programmed cell death occurs, the pH of the environment inside the cell will decrease due to increased lysosome activation [55,56]. The extracellular environment of the tumor is more acidic than that of normal cells because of hypoxia in the tumor area [57,58]. Moreover, pH also affects synthesis reaction of critical ATP at the molecular level [59,60]. The change of pH value will also bring about the change of some material properties inside the life body, such as the change of physical and chemical properties of protein, which will then affect the life process [61][62][63]. According to this, it is important to track pH change in vivo, especially in-situ real-time detection in a particular region, in order to understand the mechanisms underlying some critical functions and the and progression of specific disorders.
Based on NV centers of FND, researchers have developed a variety of pH sensors for physiological conditions. Rendler et al. [64] proposed a nanoscale pH sensor by connecting paramagnetic Gd 3+ complexes to FNDs with polymer shell via a cleavable linker ( Figure 5).  As is known, the longitudinal relaxation time T 1 of an NV center was dependent on the number of spins within the effective NV-sensing radius. The T 1 relaxation time could be quantitatively adjusted by the surrounding Gd 3+ complexes (spin labels): the more Gd 3+ complexes loaded in the surface polymer shell, the shorter was T 1 . In these ND-polymer-Gd nanosensors, Gd 3+ ions were attached on polymer shell via an aliphatic hydrazone linker. The hydrolytic cleavage would be accelerated and Gd 3+ ions detach at lower pHs. Therefore, the pH values of physiological environments were detected by reading out the change of T 1 relaxation time. It is worth noting that researchers used a chirped pulse scheme to read the T 1 relaxation time to make the measurement of T 1 not disturbed by the orientation of NV center, and realized the overall reading of all NV center signals. In principle, this scheme could monitor the gradual release of dozens of Gd 3+ complex molecules at the single particle level, which made it possible to monitor local chemical processes that occur on a very small scale (10 −22 -10 −20 mol). This ND-polymer-Gd system could operate in a fairly wide pH range with a moderate accuracy (∼0.7 pH unit). For practical measurements in cells, the accuracy of this nanosensor needed to be further increased.
Although Gd 3+ ions are frequently used to modulate the NV spin relaxometry in FND-based sensor, the potential biotoxicity still remain a restriction in its application in vivo. Moreover, the pH sensor based on polymer cleavage is usually irreversible, and it responds to pH slowly (a few minutes are needed to change the T 1 relaxation time). To solve these problems, Fujisaku et al. [65] prepared a pH nanosensor using surface-modified FNDs based on relaxation enhancement without paramagnetic agents ( Figure 6). The spin relaxation time T 1 of NV centers could be perturbed by the surface charge of the FND, which were insensitive to pH. Thus, they proposed that spin relaxation time of NV centers would correlate with environmental pH if the FND was uniformly covered with surface functional groups. Following this principle, the authors obtained a pH nanosensor by carboxylating the surface of FNDs (pK a = 4-5), in which T 1 relaxation time was significantly shortened in the pH range of 3-7(∼). Moreover, the FND coated by cysteine, with pK a of 8-11, obtained a pH dependence in the pH range of 7-11(∼). Compared with Gd 3+ modified FNDs, this sensor solved the problems of crucial synthesis conditions, irreversible detection, and long response time in pH sensing. The mechanism of this method could be extended to the detection of other species. Based on selective ligands, calcium, magnesium, potassium or heavy metal ions can be detected, which will provide an important analytical tool for a series of life activities in cells.
Raabova and her fellows also developed a pH nanosensor based on the dependence of NV center spectroscopy on surface charge changes [66]. Unlike measuring T 1 relaxation time, the authors analyzed ratio of NV -/ NV 0 which was modulated by charged molecules on the surface of FND surface. In this strategy, the FND was coated by a stimuli-responsive polymer called polyacrylic acid. The charge of the polymer shell varied with surrounding pH with physiologically relevant ranges, then alter the ratio of NV -/NV 0 states in FND. The fluorescence spectra (ratio of intensity in spectral maximum to the intensity of NV 0 ZPL) of the nano-optode showed a monotonous increase in the pH range of 5-8(∼). This work provides an inspiring readout modality of NV center in FND, which can be further used in (bio)sensing applications together with recent improvements in optical measurements of the charge of NV centers and preparative schemes for FND.
Although NV center can provide some information about pH in cellular environments, the researches in this field are still limited probably because of the complexity of the readout devices. It is also challenging to establish a universal pH transduction strategy that can be applied to various systems. Current researches are mostly focused on utilizing ND as a biocompatible host carrier to load pH-responsive substances as the signal reporter [67]. If the readout apparatus was further simplified and the detection scheme was optimized, the NV centers with high probe sensitivity and space-time resolution would be a very competitive pH sensor.

Bioactive substances
The bioactive substance changes caused by life activities are strong evidences that directly reflect the process and mechanism of the activity [68,69]. In that case, it is vital to assaying, marking and tracking some characteristic substances in the process of investigating biochemical reactions. The state of NV center can be optically and efficiently polarized and read out due to their special structural characteristics. At the same time, high-density NV centers can improve the signal-to-noise ratio and provide high spatial resolution [70,71]. Therefore, FNDs can provide small-scale and high-sensitivity detection and can capture slight changes in the environment in Figure 6. pH-metry by measuring t1 relaxation of a single fluorescent nanodiamond [65]. copyright © 2019 american chemical Society.
time. Corresponding with the unique photostability of NV center and the biocompatibility of NDs, researchers have developed a series of schemes to examine physiologically active species. The research focuses on protein labeling, free radical species detection and nerve signal monitoring.

Membrane proteins
Membrane proteins serve a range of essential tasks for organism survival. Tracking specific protein in real-time, and long-term is of great significance to understand the crucial processes in living cells [72]. According to this, Hsieh et al. [73] developed a membrane protein label by coating FNDs with a thin layer of hyperbranched polyglycerol (HPG) and modifying alkyne on the surface. By using azide-alkyne-based click chemistry, the obtained alkyne-HPGFNDs realized bio-orthogonal labeling and long-term imaging of membrane proteins on live cells (Figure 7(A)). Due to the biological orthogonal reaction, the alkyne-HPGFND can target specific membrane proteins without interfering with the normal physiological process on living cells. This study demonstrated alkyne-HPGFND particles 50 nm that were sufficiently bright to detect single particles by standard fluorescence confocal microscope. Magnetic signal modulation can effectively remove background interference and achieve fluorescence background-free detection, which is suitable for quantitative measurement. Figure 7(B-D) shows the real-time tracking of membrane protein by the fluorescent tag. The authors had also tracked the movement of integrin α5 and β1 in living cells in the short and long term.
As a significant inspiration, this work offered the possibility of using FND to continuously track target membrane proteins on living cells. The current issues in FND-based protein tracking are mostly concerned with how to label proteins with high levels of efficiency, specificity, and uniformity. Through controllable FNDs' modification, improved protein tagging methods, together with super-resolution fluorescence microscopy of NV centers, and FNDs can provide more favorable   [77]. uncoated FnDs reached to the cytosol (a1) and anti-VDac2 antibodies coated FnDs (aVDac2-FnDs) reached the mitochondria (a2). isolated mitochondria were measured before and after stimulation with carbonyl cyanide 3-chlorophenylhydrazone (cccP): catalase (cat) and superoxide dismutase (SoD). copyright © 2021 the authors, some rights reserved; exclusive licensee american association for the advancement of Science. no claim to original u.S. Government Works. Distributed under a creative commons attribution noncommercial license 4.0 (cc BY-nc). and optional means for live cell imaging in molecular biology research.

Free radical species
Free radical is a characteristic substance generated in the metabolic process of organisms. It is related to the energy production, apoptosis and aging process of organisms [74,75]. Therefore, it is necessary to develop a method for real-time monitoring of free radicals in vivo. For the first time in 2020, Martinez et al. [76] utilized the T 1 relaxation times of FNDs as indicator to determine free-radical concentration in-situ during chemical reaction. The FNDs used in this work were commercially available without additional modification process. The FND magnetometry measured a concentration of hydroxyl radical radicals as low as 2 μmol, which are naturally present in living cells. Two important OH-generating reactions in biological environmentsthe photolysis of H 2 O 2 and the Haber-Weiss reactionwere applied to demonstrate the in-situ measurement of ·OH. To simulate the biological medium, the presence of salts and proteins were taken into account, suggesting that a protein corona might affect the sensing performance. However, this work mainly focused on the detection of free radicals in vitro. In 2021, the group utilized the functionalized FNDs to detect free radical in living cells or isolated mitochondria [77]. After physically adsorbing anti-VDAC2 antibodies on FND, the aVDAC2-FNDs could target mitochondria in macrophages and detect radicals with subcellular resolution through measuring T 1 . Figure 8 briefly shows the principle of the probe targeting mitochondria. Interestingly, the measurements were nondestructive and repeatable to monitor the metabolic activity in living cells.
The peroxidase-mimicking activities of oxygenated nanodiamonds have been recently studied [78]. For the first time, Wu et al. [79] combined the catalytic activity activities of NDs with their intrinsic quantum sensing to create a self-reporting H 2 O 2 sensor (Figure 9). The oxygen-terminated FNDs acted as peroxidase, decomposing H 2 O 2 into hydroxyl radicals (·OH), then the amount of ·OH free radical could be read out by detecting the shorten of T 1 . This research demonstrated local production and quantitative detection of H 2 O 2 with molecular-level sensitivity (∼3 radicals) and nanoscale spatial resolution. Considering the important role of H 2 O 2 in various physiological processes, this FNDbased H 2 O 2 sensor offered the potential to understand of the role of H 2 O 2 in more complex biological process, such as immune response, cardiovascular diseases, and cancer.  However, the free radical determination approach based on T 1 relaxation time of NV center has inherent problems. As discussed above, T 1 is also sensitive to pH fluctuations. It remains difficult to develop FND sensors that response to a single target parameter or reduce the impact of other environmental factors. At the same time, the quantitative relation in most cases is only limited to the hydroxyl radical in aqueous media, rarely considering the influence of other interfering species on the quantitative measurement, especially in the living cells. In the future, establishing a mathematical model that is more appropriate for the real biological environment would benefit the utilization of FNDs in free radical detection.

Virus
It is crucial for disease prevention and treatment for infectious diseases caused by certain viruses to be identified in the early stages. This is especially important for diseases with high mortality and high transmission risk, like HIV or COVID-19 [80][81][82]. The primary method for detecting viruses is the PCR with reverse transcription (RT-PCR), but it requires careful primer and probe design, pretreatment procedure, specialized equipment, and trained personnel. Furthermore, since the number of viruses in the patient's body remains very low during the early stages of infection, it is necessary to develop detection methods with high sensitivity, high accuracy, and low detection limit.
Fluorescent markers have been extensively studied for the early detection of diseases, but the sensitivity is still limited by the background autofluorescence and low brightness. The NV centers in FNDs have advantages characteristics for in vitro diagnostics, including high brightness and selective manipulation by microwaves or magnetic fields. Miller et al. [83] employed FND as an ultra-sensitive tag in a lateral flow assay (LFA) format for in vitro detection of HIV virus with high sensitivity and specificity ( Figure 10). In this protocol, microwave field was applied to modulate the emission intensity, together with frequency domain analysis, separating the signal from the background autofluorescence. Combined with the widely used low-cost paper LFAs technology, a detection limit of 8.2 × 10 −19 mol was achieved for the biotin avidin binding model, 10 5 times more sensitive than that of gold nanoparticles. Single copy detection of HIV-1 RNA could be performed by adding a 10 min isothermal amplification step. It is noteworthy that the power consumption for microwave generation was relatively low (0.25 W), allowing for ultrasensitive diagnosis and monitoring in a point-of-care device given portable fluorescent readers or smartphone-based devices. This ultra-sensitive quantum diagnostic platform is applicable to a variety of diagnostic test forms and diseases, and has the potential to improve the early diagnosis of diseases.
Under the background of COVID-19 pandemic in 2019, effective and rapid diagnosis and detection of emerging new virus has become an urgent need. Li et al. [84] proposed a quantum sensor for detecting COVID-19 based on FND with NV center (Figure 11(A, B)). Previous studies have proved that attached Gd 3+ can increase the magnetic noise strength felt by NV spins and quench their T 1 time. In this theoretical model, the surface of FND was modified by a cationic polymer to adsorb the DNA strand (c-DNA) complementary to the Figure 11. overview of the diagnosis protocol [84]. (a) a sample is collected from the upper respiratory tract, e.g. with a nasopharyngeal or throat swab, followed by nucleic acid extraction. (b) test samples that might contain virus Rna are loaded into microfluidic channels containing functionalized nanodiamonds. (c) mechanism of magnetic noise quenching. copyright © 2022, american chemical Society. viral RNA, followed by binding Gd 3+ complexes (DOTA-Gd 3+ ). In the presence of viral RNA, the c-DNA-DOTA-Gd 3+ pair could be separated from the FND surface due to the hybridization of c-DNA and viral RNA. This process is shown in Figure 11(C). The detachment of Gd 3+ could result in a large change in the NV photoluminescence yield, thus the detection of virus was realized. Theoretical simulation results showed that the detection limit was as low as hundreds of viral RNA copies. The false negative rate (FNR) reached below 1%, which is far lower than the most advanced RT-PCR diagnostic method. In addition, the technology can be further promoted to diagnose other RNA viruses (like MERS) by using surface c-DNA specific to the target virus.

Summary and outlooks
The research on NV center of FNDs is still ongoing due to the unique physical and chemical characteristics. The most explored property of NV centers is their photoluminescence, which is superior valuable as a fluorescent label for imaging. Besides, current research goes deep into its electron spin manipulated by external magnetic, microwave radiation, or electric fields. These characteristics of optical readout and microwave/magnetic field spin manipulation offer potential applications in submicron magnetometers, quantum bits, quantum computers, quantum sensors, etc. Especially, in light of the excellent biocompatibility of nanodiamond, FNDs with NV centers have broad application in biosensing. Up to now, FND-based nanosensors have been developed to detect the physiological parameters (temperature, pH), bioactive substances (membrane proteins, free radical), and virus in intracellular and extracellular environments. One of the key technologies was to design transduction system responding to changes in pH/temperature or concentrations of target species while transducing signal to change of fluorescence or T 1 relaxation time of NV.
However, the sensing performance of FND biosensors is limited by the quality of diamond materials. An important requirement is to synthesize FNDs with adequately high NV center concentrations, showing brightness and sizes competitive with dyes or other fluorescent nanoparticles. Meanwhile, the components of biological system are complex, and environmental factors can affect the signal transducing of NV center-based biosensors. It is a crucial problem to increase the sensitivity of a nanosensor to the target analyte and resist the disturbance of other interfering species by modifying NDs materials or optimizing molecular transduction system. At the same time, another challenging is to develop effective signal detection methods and improve signal readout equipment based on NV center before pushing FND sensors into practical application.