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Abstract
Artificial sound enamel and caries enamel lesions were prepared. The sound enamel did not emit fluorescence in the visible spectrum. The spectrum of Zn(II)-protoporphyrin-9 biofilm from caries enamel lesions showed the fluorescence emitted by Zn(II)-protoporphyrin-9, with two main peaks at 630 and 690 nm. The luminescence properties of protoporphyrin-9 change depended on the amount of Zn(II). The increase in fluorescence intensity as Zn(II)-protoporphyrin-9 penetrated deeper to 1.75 mm was appropriate for the diagnosis of caries enamel lesions. Fluorescence intensity was maximum when Zn(II) reached 0.0256 µM and significantly produced a high contrast of fluorescence image together with high fluorescence quantum efficiency and photostability.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Porphyrins are the organic fluorophore components, which are naturally found in some oral anaerobic bacteria including Prevotella intermedia, Actinomyces naeslundii, and Actinomyces israelii, which could produce the bright-red fluorescence maxima at wavelengths of 635 and 700 nm under UV excitation. Biofilm development results in the dental plaque. The bacterial metabolism products so-called protoporphyrin-9 (PPIX) containing two carboxylic acid substituent groups, further give rise in dissolution of tooth minerals, resulting in the formation of micropores as initial-to-advanced dental caries [1,2]. PPIX fluorescence could therefore predict the maturation of microorganism in dental biofilm. The denser the bacterial colonies, the more intense the fluorescence signal will be. Practically, human dental as anisotropic medium limits distribution of this organic fluorophore, because the observed “red spots” represented PPIX accumulation is not homogeneously distributed over the surface lesions. In addition, the partial staining solution disqualifies PPIX fluorescence for the detection of smooth surface lesions. The stains also remain for some time. To help inform and improve the human dental healthcare, investigations on the developments of dental lesion screening motivate further studies on selective and sensitive substances on the existence PPIX, which deeply enhances the detection of PPIX fluorescence in the dental enamel or even in dentine.
So far, the noninvasive and sensitive imaging as well as spectroscopic methods has gained importance in biomedical applications [3]. As compared with conventional white light diffusion-based detection method, light-induced fluorescence-based detection method with exogenous organic chromophores including 5-aminolevulinic acid (5-ALA) and methyl aminolevulinate (MAL), are highly desirable. ALA-PPIX with the high-dose of 0.15 mM ALA emits the PPIX fluorescence in the meningioma [4] and in the low-to-high grade glioma [5], whereas MAL-PPIX at the same dose emits the PPIX fluorescence in the superficial basal cell carcinoma [6]. However, inorganic-induced PPIX with the low-dose to avoid any adverse effects has not been exploited especially for detection of the caries enamel lesions. In addition, there is another factor served as the fundamental limitation of the light-induced fluorescence-based technique. Since the Soret-absorption peak of PPIX (400-500 nm) is 20-30-fold larger as compared to Q-absorption band (500-750 nm), a blue light irradiation within Sorbet band is practically preferred for high PPIX absorption. However, it induces the PPIX photodecomposition and has a poor penetration depth into the human dental. Thus, based on the light propagation theory, the light scattering of human dental affects the spectra for both fluorescence and diffuse reflectance with respect to path lengths of the collected photons. As such, the measured PPIX fluorescence in human dental is a modulated spectrum, which does not trivially reflect the true concentration of PPIX. One solution is to bound PPIX with the high-diffusible Zn(II) to observe the caries generally bound in the enamel layer of 1-2 mm. The red light and fluorescence radiation are therefore less absorbed and scattered by the enamel caries. In this case, the influence of scattering properties of sample and penetration depth could be neglected at 1-2 mm from the surface imaging system.
To the best of our knowledge, notwithstanding Zn(II) has wide applications in biomedical science [7]; their applications in dentistry are unknown. Zn(II) are therefore proposed as a suitable candidate detection probe in the imaging-guided caries lesions, owing to their strong formation with PPIX in the visible region and high reactivity towards pathogenesis responses. Irradiation Zn(II)-PPIX complexes under UV light would produce the strong and stable emission in the red wavelength regime. Zn(II)-PPIX complexes is also possible to improve the fluorescence intensity, quantum yield, and photostability.
Herein, we chose the beta-tricalcium phosphate to mimic the matter composition of dental enamel, whereas PPIX as a fluorescence medium was utilized to mimic formation of dental biofilm and microbial deposition. Caries lesions classification were controlled through PPIX content along with their underlying mechanism of action, through application of both qualitative (imaging and color) and quantitative (spectroscopy) methods. Fluorescence-based test would confirm the selective and sensitive combination of Zn(II) into the PPIX biofilm-enamel model as an ideal detection probe. Fluorescence characteristics between the healthy enamel model and Zn(II)-PPIX biofilm + enamel model were clearly differentiated. The limit of detection of Zn(II)-PPIX biofilm in the enamel model was finally determined.
This study is novel because of four reasons. One, PPIX bound with non-organic Zn(II) is used as a novel material and fluorescence probe, achieving the enhance fluorescence for caries observation in the human enamel layer. Two, in comparison to the current organic-induced protoporphyrin-9, the remaining Zn(II) is easily removed through oral rinse after clinical diagnosis and treatment of the caries enamel lesions. Three, our observation is qualitative and quantitative analysis of the caries enamel lesions with help of fluorescence method in place of illumination method, such as digital imaging of fiber optic transillumination. Four, it is possible that on-line Zn(II)-protoporphyrin-9 autofluorescence measurement during the phototreatment would give the readers gain insight into the efficiency of photodynamic therapy in dentistry in the future.
2. Experimental
2.1 Materials
Polymethylmethacrylate (PMMA), zinc ions (Zn(II)), beta-tricalcium phosphate ( β-TCP, Ca3(PO4)2), protoporphyrin disodium salt (PPIX, C34H32N4Na2O4), dichoromethane (DCM), and dimethyl sulfoxide (DMSO), were purchased from Sigma Aldrich, USA.
2.2 Preparation of the dental enamel model
To obtain the dental enamel model, 100 mg of β-TCP was diluted by 1 mL of DMSO and hence sonicated under ultrasonic bath (Model: CMT-50, USA) for 30 min at a room temperature of 25°C according to previous protocol [8]. The prepared solution was added with 200 mg of PMMA in 1 mL of DCM under vortex mixer for 2 min. The 1 mL of colloidal dispersion was dropped on a clean glass slide, using a homemade mold design to increase the evaporation time up to 1 hr. A typical phenomenon related to drying upon drop casting deposition so-called coffee ring effect would induce the accumulation of colloidal suspension at the edge of the drying solvent. As a result, dental enamel model close to the dried edge typically contained the thicker layers of β-TCP, whereas the dental enamel model around the center was composed of thinner layers of β-TCP.
2.3 Preparation of the PPIX biofilm + enamel model
The 10 μM of stock solution of PPIX was diluted by DMSO and added with 200 mg of PMMA in 1 mL DCM under vortex mixer for 2 min (Model: GENIE 2; G560E Scientific Industries, USA). PPIX biofilm model was set at 2.5 μM to be close to in vivo concentration, onto the enamel model [9].
2.4 Preparation of the Zn(II)-PPIX biofilm + enamel model
One of important applications of the fluorescence detection probe was as a fluorescence enhancer, which was normally realized by introducing the artificial defects into a prepared PPIX-biofilm + enamel model. Due to high requirement of fluorescence imaging efficiency in dental diagnostic and treatment, Zn(II) was sufficiently sensitive to the line and point defects. In this respect, 0.82 M of stock solution of Zn(II) was diluted by deionized water. Zn(II) was set no greater than 2.5 mg/mL similar to ZnO, which was safe for normal tissues [10]. After that, Zn(II) was fabricated the line by line or even point by point defects through a drop casting deposition onto PPIX-biofilm + enamel model. The dimension of specimen was 10 × 10 mm2 with different thicknesses of biofilm (0.50, 0.75, 1.00, 1.25, 1.50, and 1.75 mm). If a total thickness of the specimen was maintained at 2.00 mm, the remaining enamel thickness was 1.50, 1.25, 1.00, 0.75, 0.50, and 0.25 mm, respectively. Cocktail sample solution expressed as µM was detailed in Table 1 and effect of geometrical parameters on the Zn(II)-PPIX biofilm + enamel model was detailed in Table 2. After 90 min of incubation period, maximum image contrast of each specimen was reached and ready for all tests of the fluorescence intensity and color.
Table 1. Optical properties and color analysis of 2.50 μM of PPIX-biofilm with difference concentrations of Zn(II). Ratio of thickness of Zn(II)-PPIX biofilm and enamel model is 1 mm:1mm.
Table 2. Optical properties and color analysis of 2.50 μM of PPIX-biofilm with different ratio of thickness of Zn(II)-PPIX biofilm and enamel model. Zn(II) concentration is 0.0256 μM.
2.5 Fluorescence techniques
White light from a tungsten lamp (Model: Megalight-50Hoya-Schott, Japan) was incident on the Zn(II)-PPIX biofilm + enamel model so that the absorbance spectra were detected with UV-Vis spectrometer (Model: AvaSpec-ULS2048 Avantes, USA). After that, UV light was used in studying the component of fluorescence band of the specimen. The UV light with the excitation wavelength of 400 nm, which corresponded to the excitation wavelength of PPIX, was incident at a right angle on the specimen. Fluorescence maximum at wavelength of 630-660 nm of Zn(II)-PPIX biofilm + enamel model as compared with the standard dental enamel model (control) was detected and recorded by the same spectrometer. The experiments were conducted in triplicate. All data were analyzed by one-way ANOVA test (p<0.001). All specimens had the same color at the start of the interventions to mimic the human dental.
Dependence of the peak fluorescence intensity on Zn(II)-PPIX biofilm + enamel model prepared at five thickness ratios of Zn(II)-PPIX biofilm and enamel model, 0.33, 0.60, 1.00, 3.00, and 7.00, was carried out. A polynomial plot was injected in triplicate. The resulting fluorescence peak (y) versus thickness ratio (x) was hence plotted and treated by least squares method of non-linear regression for determination of the limit of detection (LOD) according to Ref. [11].
If we defined that QREF is the quantum yield of the reference PPIX, n is the refractive index of the biofilm, I and A are the integrated fluorescence intensity and absorbance peak, respectively, the fluorescence quantum yield of Zn(II)-PPIX biofilm + enamel model (Q) is calculated by [11]
All specimens were kept in dark to avoid 400 nm excitation from the ambient light prior to detect the fluorescence imaging (Model: Nikon eclipse Ti5, Japan). The specimen was excited under UV light (Model: Nikon C-HGFI, Japan). A customized optical filter was designed to match the excitation and emission profiles of PPIX molecules. A charge-coupled device camera subsequently captured the high contrast fluorescence image emitted by Zn(II)-PPIX biofilm + enamel model. If NA is the numerical aperture and λ is the wavelength, the resolution of fluorescence imaging (R) is calculated by [12]
Substituting NA = 0.95 and λ = 400 nm into Eq. (2), the resolution of this method was 210 nm.
2.6 Colorimetric technique
Coloration of Zn(II)-PPIX biofilm + enamel model was identified by a colorimeter (Model: NR200 3NH, Hong Kong). According to CIE L*a*b* system, three-coordinate value (L*a*b*) was present, in which L* axis demonstrates the degree of lightness/darkness in a range of 0-100 (black-to-white), a* axis represents the angle of green/red, and b* axis refers between blue and yellow. The baseline of color is measured over the standard dental enamel model (control). The degree of color variation is calculated via the variations of the three values: ΔL, Δa, and Δb, as [8]
3. Results
3.1 Fluorescence spectra
Zn(II)-PPIX biofilm + enamel model shows a strong Sorbet centered at 400 nm with varying the concentrations of Zn(II) as shown in Fig. 1. The spectrum of pure PPIX is employed as a reference system. The absorbance of all samples is proportional to the concentration of Zn(II) according to Beer-Lambert’s law [11]. In the fluorescence spectrum as seen in Fig. 1(a), a blue shift of about 30 nm occurs due to the increase of Zn(II) ions from Sample# A to C. This shift is due to the entry of Zn(II) into the porphine core of PPIX and their binding [13]. This binding increases the quantum efficiency of the fluorescence of each PPIX molecule, which is responsible for the increase in the fluorescence intensity of sample# C. However, after 0.0256 µM of Zn(II), the binding saturates, and Zn(II) ions begin to be present in the enamel. The increase in absorbance from sample# D to F might be due to this increase in Zn(II) ions. The absorption of excitation light by these Zn(II) ions reduces the number of photons available to excite the bound Zn(II)-PPIX. It is therefore considered that the number of photons decrease from sample# D to F in the fluorescence spectrum. The sharp edge on the short wavelength side at 500 nm is caused by the cut-off below 500 nm of the optical long pass filter in front of the UV-Vis spectrometer. In this regard, the measured quantum yield is 0.0447-0.1566 when a ratio of thickness of Zn(II)-PPIX biofilm and enamel model is maintained at 1 mm:1 mm. The optimal concentration ratio of Zn(II) and PPIX is 0.0256:2.5 = 1:100. With increase in Zn(II)-PPIX biofilm:enamel thickness ratio from 0.33:1 to 7.00:1 by keeping the amount of Zn(II) and PPIX constant as shown in Fig. 1(b), a periodic enhancements of the absorption and fluorescence intensities are observed, whereas the fluorescence quantum yield of Sample# C1-C6 is significantly improved at 0.1216-0.2244, >3 times greater than that of Sample# A and B. These ratios are important parts for detecting the enamel caries and caries lesion depth is able to estimate in the diagnostic process. During the caries decay process under acidic conditions (pH=6.60), β-TCP in the enamel at biofilm-enamel interface is partially dissolved to form Ca2+ ions [14]. These ions tend to infiltrate through the entire depth of Zn(II)-PPIX with increasing biofilm thickness [15]. Therefore, it is possible that the presence of free calcium ions together with Zn(II)-PPIX leads to increase in the fluorescence intensity from sample# C1 to C6 [16].
Fig. 1. (a) Correlation between the absorption and fluorescence emission spectra of Zn(II)-PPIX biofilm + enamel model to stepwise addition of Zn(II). Spectrum of PPIX alone is used as a reference system. Zn(II)-PPIX has two peaks in the absorption spectra. The blue band is markedly larger as compared to the absorption band at 500-550 nm. Excitation is provided near wavelength of 400 nm. A, B, C, D, E, and F represent 0.0064, 0.0128, 0.0256, 0.0513, 0.1025 and 0.2050μM of Zn(II), respectively with constant concentration of 2.50 μM of PPIX. (b) Absorbance and fluorescence spectroscopic responses of Zn(II)-PPIX biofilm + enamel model to stepwise of addition of Zn(II)-PPIX biofilm thickness. C1, C2, C3, C4, C5 and C6 represent thickness ratio of Zn(II)-PPIX biofilm:(enamel = 1), 0.33, 0.60, 1.00, 1.67, 3.00, and 7.00, respectively, with constant content of Zn(II) = 0.0256 μM and PPIX = 2.50 μM. Total thickness of specimen is kept constant at 2 mm.
3.2 Fluorescence imaging
The dotted lines are a boundary between the enamel model and Zn(II)-PPIX biofilm model observed from the z-direction. Dental enamel model and Zn(II)-PPIX biofilm + enamel model observed in the upper (z) direction show the different red fluorescence colors under UV excitation as seen in Fig. 2(a) with their corresponding modeling geometry as seen in Fig. 2(b). It is noted that width, length, and thickness of the samples that mimic the enamel in the x-, y-, and z-direction are 10, 10, and 0-2 mm, respectively. Dental enamel model does not emit any fluorescence color on a white background. However, it is seen that there is a relationship between Zn(II)-PPIX fluorescence color and Zn(II)-PPIX biofilm thickness. There is an increase in the red fluorescence color as the thickness of sample increases from Sample# C1 to C6 and it is maximal at Sample# C6, conforming to the results from the fluorescence intensity in Fig. 1(b). In this respect, caries enamel lesions are therefore divided into two sets for the assessment of sensitivity and selectivity. That is, Zn(II)-PPIX biofilm + enamel model of Sample# C1-C3 illuminates the weaker red fluorescence than that of Sample# C4-C6, due to the presence of Zn(II)-PPIX fluorophore with smaller density. Therefore, Zn(II)-PPIX biofilm + enamel model with Zn(II)-PPIX biofilm: enamel thickness ratio of less than 1 (C1 and C2) is classified as the benign-like enamel lesions, whereas Zn(II)-PPIX biofilm + enamel model with biofilm: enamel thickness ratio of higher than 1 (C4-C6) is classified as the malignant-like enamel lesions.
Fig. 2. (a) Fluorescence imaging of the enamel model (control) and Zn(II)-PPIX biofilm + enamel model observed from the z-direction. The dotted lines are a boundary between the enamel model and Zn(II)-PPIX biofilm model. (b) Modeling geometry of the enamel model and Zn(II)-PPIX biofilm + enamel model. Width, length, and thickness of the samples that mimic the enamel in the x-, y-, and z-direction are 10, 10, and 0-2 mm, respectively.
4. Discussion
4.1 Limit of detection
Fluorescence peak intensity versus the ratio of Zn(II)-PPIX biofilm and enamel model at a given concentration of Zn(II) (0.0256 µM) and PPIX (2.5 µM) indicates a non-linear regression line for the caries enamel lesions characteristics (Fig. 3). There is an increase in the fluorescence intensity as the ratio of biofilm to enamel model increases from Sample# C1 to C6 and it is maximal at Sample# C6. The optimal concentration ratio of Zn(II) and PPIX is 0.0256:2.5 = 1:100. The concentration of ZnO at this level is suggested no effect on the real dental enamel as long as it does not go over 0.15-3.06 mM [10,17]. The dose of Zn(II) under our study is also smaller than that of ALA and MAL [4–6]. Although the experimental model is simplified and controlled to refrain from interferences, the real human enamel specimen would be carried out to verify the relevance in a broad clinical future view. Nonlinear least–squares fit of major (y1) and minor peaks (y2) of fluorescence of the specimens are estimated as y1 = -10.6930x + 63.5829 and y2 = -10.6400 + 29.8430, respectively, where x is Zn(II)-PPIX biofilm: enamel thickness ratio. Substituting uncertainty constant (k = 3), slope (a = 10.6930 for y1 and 10.6400 for y2), SD (1.4268 for y1 and 1.3125 for y2), n = 3, and readout resolution (0.01), the detection limit for y1 and y2 are 0.2311 and 0.2137, respectively. Mean value of the limit of detection is therefore used for prediction of caries enamel lesions of no later than 0.25 mm in depth above 1.75 mm the dental enamel model (total thickness of specimen is kept constant at 2.00 mm). The layer of dental enamel is kept constant at 2.00 mm with two reasons. One, based on the acidogenic theory (Miller’s theory) of dental caries, there is the interplay between the mineral contents of the dental enamel and dental caries. Enamel layer acts as a filtering membrane allowing the transition of any substances from the exterior to the interior. Enamel contains about 4% water and organic molecules, allowing the flow of PPIX, and then giving rise to disintegration of the organic matter and posteriorly conditioning demineralization of β-TCP component. These enamel areas with disintegration of the organic material, the existence of superficial cavitation, and large structural defect such as cracks, which are rich in organic material, could facilitate the penetration of PPIX into deep areas of the dental enamel [18]. Two, since the light fluence rate at the end of enamel layer is over 95% of the value on the enamel surface, at the depth of 1-2 mm, the excited light could completely penetrate into the enamel model’s area for stimulation, whereas the emission light could escape to the enamel surface for the fluorescence detection. However, the photons are almost completely absorbed at the depth of 3-7 mm. After absorbing the excited light, PPIX fluorophore makes fluorescence photons more possible to propagate uniformly and multi-directionally. PPIX emits more fluorescence photon along a given depth in the dental enamel. The light multiple scattering is a main optical parameter in determining the light propagation and fluorescence distribution inside the dental enamel (thickness = 1-2 mm) and dentin (thickness = 3-7 mm) [19]. According to the scattering coefficient of β-TCP in enamel (μs = 5–25 mm-1) and tubules in dentine (μs = 100–140 mm-1) [20], the photon density of both diffusion transportation of excited light (400 nm) and following intrinsic fluorescence (630-660 nm) tend to extensive decay with the depth increment corresponding to the increase of scattering coefficient from dental enamel to dentine. Therefore, this test might fail at depths >2 mm, in part due to loss of the aforementioned fluorescence signal. The performance might be significantly enhanced using the stronger light source, such as high-power laser, and more sensitive camera, such as electron-multiplying CCD. Both options are currently being pursued.
Fig. 3. Non-linear regression line for prediction of location and boundary of caries enamel lesions from Zn(II)-PPIX biofilm + enamel model. The fluorescence intensity is observed in the same optical configuration as in Fig. 2 (z-direction). The vertical axis shows the fluorescence intensity, the horizontal axis shows the ratio of biofilm to enamel model, and the orange and blue plots show the fluorescence intensity of the major and minor peaks at each ratio of biofilm to enamel model, respectively. The orange point curve and the blue point curve are the least square fitting of the major and minor peak fluorescence intensities, respectively.
The potential impact of Zn(II)-PPIX photodecompsotion is also tested. It is verified that the total signal loss due to photodecompsition during the selected integration time (10-500 ms, a step of 10 ms) in photostability of Zn(II)-PPIX response is in any case below 10%. Assuming that the actual measurement period (when Zn(II) in contact with the PPIX biofilm-enamel model) does not have to be longer than 1 sec to get a sufficient fluorescence signal. Therefore, photodecomposition should not be a limiting factor, even if repeated light exposure is considered.
4.2 Fluorescence imaging
In Fig. 4(a), a gray value in a range of 0-50 (darkness-to-brightness) represents the intensity or brightness of the samples. This intensity distribution is observed in the same optical configuration as in Fig. 2 (z-direction). The dotted lines are confirmed as a boundary between the enamel model and Zn(II)-PPIX biofilm model as illustrated in Fig. 2(a). Notably high emission intensity at wavelength of 630 nm under UV excitation is correlated with Zn(II)-PPIX distribution in the UV-illuminated area of Zn(II)-PPIX biofilm + enamel model. In the human enamel layer of 1-2 mm, it is possible that the exogenous application of 0.0256 µM of Zn(II) solution to human dental (by several routes, such as instillation and ointment), induces an enhancement of fluorescence intensity, leading to the high subjective fluorescence image contrast. Zn(II) presumably further facilitates the transportation of PPIX through the diffusion and selective accumulation, into the enamel model.
Fig. 4. (a) Gray value of Zn(II)-PPIX biofilm + enamel model in a range of 0-50 (darkness-to-brightness) represents the intensity or brightness of the samples. This intensity distribution is observed in the same optical configuration as in Fig. 2 (z-direction). The dotted lines are confirmed as a boundary between the enamel model and Zn(II)-PPIX biofilm model as illustrated in Fig. 2(a). (b) Fluorescence image contrast and the degree of color variation (ΔE*) of Zn(II)-PPIX biofilm + enamel model.s
Stokes shift (30 nm) of Zn(II)-PPIX biofilm + enamel model is a great advantage for fluorescence imaging, because a small Stokes shift causes a small measurement error due to noise from the excitation and scattered light. Since Zn(II)-PPIX biofilm + enamel model emits at relatively long wavelength, overlapping caused by absorption and autofluorescence by PPIX molecules is reduced, potentially facilitating the transportation of PPIX fluorescence at deeper penetration in the enamel model.
In Fig. 4(b), with increased accumulation of Zn(II)-PPIX complexes, this test improves the degree of color variation (ΔE*) correlated with the fluorescence image contrast. The better the image contrast caused by Zn(II)-PPIX biofilm + enamel model, the more detailed information about discriminating different sample structures between enamel model (control) and Zn(II)-PPIX biofilm + enamel model are gained.
5. Conclusions
Application of both qualitative (fluorescence imaging) and quantitative (UV-Vis spectroscopy and colorimeter) methods offered a full understanding about the fluorescence enhancement of caries enamel lesions through Zn(II)-PPIX biofilm + enamel model. When Zn(II)-selective binding units were attached to PPIX biofilm-enamel model, the developed PPIX biofilm-enamel model showed the unique optical performance characteristic of Zn(II)-PPIX biofilm + enamel model in a comparison to a standard enamel model. Under the UV light source, fluorescence spectra of PPIX biofilm-enamel model at the different concentrations of Zn(II) were measured and the optimum dose of Zn(II) was then determined. Fluorescence maxima at wavelengths of 630 and 690 nm corresponded to the emission peaks of Zn(II)-PPIX complexes. Fluorescence imaging experiments showed that Zn(II) generated the excellent image contrast of the lesions-to-healthy enamel through fluorescence activation in PPIX-biofilm sites, which offered the information of location and boundary of caries enamel lesions. Our fluorescence detection probe confirmed a potential strategy for imaging-guided dental caries in Zn(II)-PPIX biofilm + enamel model.
Funding
King Mongkut's Institute of Technology Ladkrabang (2564-02-05-016).
Acknowledgments
The authors thank Prof. Pattareeya Damrongsak from the King Mongkut's Institute of Technology Ladkrabang for providing some instruments. This research was funded by King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand (grant number 2564-02-05-016).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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