A Label-Free Fluorescence Polarization for Protein Detection Based on Albumin Nanoparticles

Abstract

Human serum albumin (HSA), an endogenous protein, was employed in the synthesis of nanoparticles. The treatment of an HSA solution with ethanol and glutaraldehyde resulted in the formation of human serum albumin nanoparticles (HSA NPs), which exhibited a weak fluorescence emission peak at 515 nm upon excitation at 360 nm. Importantly, these synthesized HSA NPs displayed a pronounced fluorescence polarization (FP) signal under identical excitation and emission conditions. Furthermore, incubation of the HSA NPs with specific DNA aptamers targeting lysozyme and thrombin led to a significant decrease in the FP values. This reduction in FP was effectively reversed upon the addition of lysozyme and thrombin. Based on these observations, a label-free fluorescence polarization-based detection platform for lysozyme and thrombin was developed utilizing HSA NPs and a DNA aptamer system.

1. Introduction

Fluorescence polarization (FP) is an analytical method that leverages the relationship between molecular rotational behavior and the polarization characteristics of fluorescence emission [1]. This technique is widely applied across multiple fields, such as the study of molecular interactions, drug development, and immunoassays [2]. When fluorescently labeled molecules are excited with plane-polarized light, those exhibiting limited mobility emit fluorescence that preserves the original polarization orientation. In contrast, rapid molecular rotation during the excited state results in depolarization of the emitted fluorescence [3]. Specifically, fast rotational movements of fluorophores cause the polarization direction of the emitted light to become randomized, leading to a reduction in the FP value. The rotational speed of molecules is primarily determined by their volume. The FP value is determined by measuring fluorescence intensity at the vertical (F_v) and horizontal (F_h) axes, calculated using the following equation:

FP (mP) = 1000 × (F_v − G × F_h)/(F_v + G × F_h)

(1)

FP values are expressed in millipolarization units (mP), and G represents the G correction factor [4,5].

According to the principle of FP, fluorescently labeled small-molecule antigens compete with sample antigens for antibody-binding sites. The formation of antigen–antibody complexes leads to a reduction in molecular rotation, thereby causing an increase in the FP value. Similarly, fluorescently labeled DNA or RNA aptamers bind to their targets, which restricts molecular rotation and consequently elevates the FP value. These changes in FP value can be quantitatively monitored and utilized for the detection of proteins and molecules [6,7]. For example, Zhang et al. employed graphene oxide nanosheets to enhance the FP signal of carbon dot-labeled lactoferrin aptamers. Their study demonstrated the feasibility of detecting tear lactoferrin within a concentration range of 0.66–3.32 mg/mL based on the decrease in FP values [8]. Jia and colleagues investigated the FP alterations of aptamer-modified Ag₁₀ nanoparticles and dye-labeled DNA sequences following the introduction of polychlorinated biphenyls [9]. Zhang et al. established an aptamer-based FP assay for the quantification of exosomes, enabling quantitative analysis within a concentration range of 5 × 10²–5 × 10⁵ particles per microliter for exosomes derived from cell lines [10]. So far, most reported FP assays rely on fluorescent probe labeled aptamers or antibody [11,12,13,14,15,16].

The labeling process not only increases the cost of synthesis but may also reduce the binding affinity to the target molecule. Therefore, the development of label-free FP detection technologies is critically important to enhance the practical applicability of FP methodologies. In 2025, Wang et al. first reported the amplification of FP signals using bovine serum albumin nanoparticles induced by manganese dioxide nanosheets and further demonstrated the FP-based detection of glucose and choline in the presence of glucose oxidase and choline oxidase, respectively [17]. Lysozyme (LYZ) exhibits strong antimicrobial activity by specifically hydrolyzing the β-1,4-glycosidic bonds within the peptidoglycan layer of bacterial cell walls [18,19]. Moreover, LYZ can interfere with viral replication by forming complexes with nucleic acids [20,21]. Thrombin (TBA), a serine protease, plays a pivotal role in fibrinogen degradation, platelet aggregation, and cellular repair processes [22,23,24]. Both LYZ and TBA have been identified as important biomarkers for a variety of diseases.

In this study, human serum albumin (HSA) was used as the precursor material to prepare human serum albumin nanoparticles (HSA NPs), which exhibited a pronounced FP signal. The effects of various DNA aptamers, including an anti-LYZ sequence (DNA-1) and anti-TBA aptamers (DNA-2 and DNA-3) on the FP signal of HSA NPs, were systematically investigated. A marked reduction in the FP signal was observed upon incubation with these aptamers. Considering the established affinities of these aptamers for LYZ and TBA, the potential application of FP-based detection for these proteins was further examined by conjugating HSA NPs with the corresponding aptamers.

2. Materials and Methods

2.1. Materials

HSA, absolute ethyl alcohol, glutaraldehyde, LYZ, and TBA were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). DNA-1 sequence (5′-ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-3′), DNA-2 sequence (5′-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′), and DNA-3 sequence (5′-GGT TGG TGT GGT TGG-3′) were purchased from Beijing Dingguo Biological Reagent Company, (Beijing, China). All the reagents were of at least chemical grade. The resistivity of deionized water used in the experiment was not less than 18 MΩ·cm.

2.2. Preparation of HSA NPs

The preparation procedure was carried out in accordance with established protocols, with specific modifications as described in reference [25]. Initially, 120 mg of HSA powder was dissolved in 4 mL of deionized water. Subsequently, 8 mL of absolute ethanol was added dropwise to the HSA solution under continuous stirring for 15 min. Thereafter, 20 μL of a 50% (w/w) glutaraldehyde solution was rapidly introduced into the mixture, which was then stirred for an additional 6 h. The resulting HSA NPs precipitate was collected by centrifugation at 12,000 rpm for 15 min and redispersed in 12 mL of deionized water. This process was repeated twice, after which the collected HSA NPs precipitate was subjected to lyophilization. The purified HSA NPs powder was subsequently stored at −4 °C.

2.3. The Investigation of FP Changes in HSA NPs Incubated with DNA Sequences

The prepared DNA sequence solution was heated to 75 °C for 10 min and allowed to cool down on ice. The heating and cooling operations were used to minimize the potential misfolding of DNA sequences prior to incubation with the protein target.

A volume of 200 μL of HSA NPs solution at a concentration of 0.325 mg/mL, varying volumes of DNA sequence solution (25 μg/mL), and 1 mL of Tris-HCl buffer solution (pH 7.4, 10 mmol/L) containing 20 mM NaCl and 20 mM MgCl₂ were combined in a centrifuge tube. The mixture was then diluted to a final volume of 2 mL with deionized water and subjected to continuous agitation for 20 min at ambient temperature. Subsequently, the FP values, expressed in mP, of the resulting solution were determined using an excitation wavelength of 360 nm and an emission wavelength of 515 nm. All fluorescent tests were performed on the same equipment fluorescence spectrophotometer, Cary Eclipse, and the G correction factor was set to 1.0.

2.4. The Effect of LYZ on HSA NPs/DNA-1 System

A volume of 200 μL of HSA NPs solution (0.325 mg/mL), 200 μL of DNA-1 solution (25 μg/mL), varying volumes of LYZ solution, and 1 mL of Tris-HCl buffer (pH 7.4, 10 mmol/L) containing 20 mM NaCl and 20 m MgCl₂ were mixed in a centrifuge tube. The mixture was then diluted to a final volume of 2 mL with deionized water and subjected to continuous agitation for 20 min at ambient temperature. Subsequently, the FP values of the resulting solution were determined using an excitation wavelength of 360 nm and an emission wavelength of 515 nm.

2.5. The Effect of TBA on HSA NPs/DNA-2 System or HSA NPs/DNA-3

A volume of 200 μL of HSA NPs solution (0.325 mg/mL), 200 μL of DNA-2 or DNA-3 solution (25 μg/mL), varying volumes of TBA solution, and 1 mL of Tris-HCl buffer (pH 7.4, 10 mmol/L) containing 20 mM NaCl and 20 m MgCl₂ were mixed in a centrifuge tube. The mixture was then diluted to a final volume of 2 mL with deionized water and subjected to continuous agitation for 20 min at ambient temperature. Subsequently, the FP values of the resulting solution were determined using an excitation wavelength of 360 nm and an emission wavelength of 515 nm.

3. Results and Discussions

The denaturation of HSA was performed in an alcohol–water mixture, followed by cross-linking via the addition of glutaraldehyde. Figure 1A illustrates that the synthesized HSA NPs exhibited a characteristic fluorescence emission peak at 515 nm, along with a broad absorption band ranging from 300 to 600 nm. The morphology and particle size of the HSA NPs were further characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). As shown in Figure 1B,C, the HSA NPs appeared as aggregated spherical structures, with an average diameter of 182 nm as measured by DLS, and a zeta potential of −30.2 mV. Fourier transform infrared spectroscopy (FTIR) was utilized to investigate changes in functional groups. The FTIR spectra presented in Figure 1D for both native HSA and HSA NPs indicate a notable increase in the C–H stretching vibration at 2960 cm⁻¹ and the Schiff base (C = N) at 1580 cm⁻¹ in the HSA NPs.

Figure 2A presents the series of fluorescence emission spectra of HSA NPs under varying excitation wavelengths. Notably, the emission peak exhibits a pronounced redshift from 515 nm to 634 nm as the excitation wavelength increases from 360 nm to 460 nm. Subsequently, the FP values of HSA NPs were examined across different excitation (EX) and emission (EM) wavelength pairs. As illustrated in Figure 2B, the FP values measured at various EX/EM pairs of 360 nm/515 nm, 380 nm/538 nm, 400 nm/574 nm, 420 nm/609 nm, 440 nm/621 nm, and 460 nm/634 nm remained consistently stable.

To elucidate the origin of the fluorescence observed in HSA NPs, it is important to consider that intrinsic protein fluorescence typically arises from specific amino acid chromophores, including phenylalanine (Phe), tryptophan (Try), tyrosine (Tyr), and histidine (His) [26]. To investigate this, the synthesis of nanoparticles was conducted by substituting HSA powder with these individual amino acids. As depicted in Figure 2C, the solution prepared with His as the precursor exhibited weak fluorescence centered around 515 nm, resembling that of HSA NPs, whereas the solution derived from Try displayed a pronounced fluorescence around 450 nm. To exclude the potential contribution of glutaraldehyde, fluorescence emission spectra were also recorded for samples synthesized solely with glutaraldehyde. The results, shown in Figure 2C, indicate that glutaraldehyde alone produces negligible fluorescence within the 400–650 nm range. Collectively, these findings suggest that the fluorescence emission peak of HSA NPs primarily originates from the cross-linking interactions among glutaraldehyde, His, and Try residues.

Furthermore, various proteins, including hemoglobin, LYZ, pepsin, and trypsin, were used to replace HSA in the synthesis process of HSA NPs under identical conditions. As demonstrated in Figure 2D, hemoglobin, LYZ, pepsin, and trypsin as raw materials cannot produce remarkable fluorescence, and only HSA as raw material exhibited significant fluorescence emission around 515 nm.

As depicted in Figure 3A, the prepared HSA NPs solution exhibited a pronounced FP signal of approximately 886 mP, which remained stable over a duration of 30 min. In contrast, upon incubation with 2.5 μg/mL of DNA-1, the FP value of the HSA NPs solution demonstrated a gradual decline during the initial 15 min, after which it reached a plateau. Figure 3B,C illustrate the effects of varying DNA-1 concentrations on the HSA NPs. Specifically, Figure 3B reveals a significant decrease in the FP value of the HSA NPs solution as the concentration of DNA-1 increased from 0 to 2.5 μg/mL. Conversely, the fluorescence emission spectra of the HSA NPs solution exhibited minimal changes with increasing DNA-1 concentrations, as shown in Figure 3C. It is well established that the FP signal of a fluorogen is correlated with its molecular volume and vibrational dynamics. The hydrated particle size and zeta potential of the HSA NPs solution following incubation with 2.5 μg/mL DNA-1 (referred to as HSA NPs/DNA-1) are presented in Figure 4A. Notably, the mean particle size of the HSA NPs decreased to approximately 17 nm post-incubation, accompanied by an increase in surface negative charge to −47.3 mV. TEM was employed to confirm morphological changes subsequent to DNA-1 addition. As illustrated in Figure 4B, the HSA NPs retained a spherical and monodisperse morphology. Based on these observations, it is hypothesized that HSA NPs adsorb DNA-1 via hydrogen bonding and π–π stacking interactions [27,28]. This adsorption process substantially inhibits aggregation of HSA NPs, leading to a reduction in hydrated particle size and a decrease in FP values. Furthermore, fluorescence lifetime measurements of both HSA NPs and HSA NPs/DNA-1 were conducted. As shown in Figure 4C,D, the fluorescence decay curves can be modeled by the following equation:

I_(t) = A₁exp(−t/τ₁) + A₂exp(−t/τ₂)

(2)

The relevant parameters are offered in

Table 1. The relative parameters of the fitted curve of the HSA NP solution and the HSA NPs/DNA-1 solution.

	A1	τ1	A2	τ2	R2
HSA NPs	1090.27	1.43	39.57	13.11	0.989
HSA NPs/DNA-1	1086.85	1.38	45.07	12.44	0.991

. The fluorescence lifetime τ of HSA NPs and HSA NPs/DNA-1 was 4.35 ns and 4.39 ns, calculated by the following equation:

τ = (A₁τ₁² + A₂τ₂²)/(A₁τ₁ + A₂τ₂)

(3)

Although the FP value of HSA NPs significantly decreased after the incubation with DNA-1, the fluorescence lifetime τ of HSA NPs changed little after the addition of DNA-1.

Previous research has established that the aptamer DNA-1 exhibits a strong affinity for LYZ, with a dissociation constant (Kd) of 31 nM, which is lower than that observed for serum albumin [29,30,31,32,33]. Based on this finding, we posited that DNA-1 preferentially bound to LYZ rather than to HSA NPs. To investigate this hypothesis, we analyzed the FP changes in HSA NPs incubated with 2.5 μg/mL of DNA-1 in the presence of varying concentrations of LYZ. As depicted in Figure 5A, the FP values of the HSA NPs/DNA-1 complex increased progressively as the concentration of LYZ (C_Lyz) was raised from 0 to 8.334 μg/mL. Moreover, within the concentration range from 0 to 0.417 μg/mL, the relationship between the FP values and C_Lyz was accurately characterized by the following equations:

FP = 284.9 + 302.5 C_Lyz

(4)

The fitting coefficient R² was 0.997, and the detection limit for LYZ was 0.032 μg/mL according to the 3 s/k criterion. The relationship between the FP value and C_Lyz in the range of 0.417–8.334 μg/mL can be well described by the following equations:

FP = 391.3 + 39.6 CLyz

(5)

The fitting coefficient R² was 0.998. Instead, the fluorescence emission spectra of HSA NPs/DNA-1 system changed a little with the increase of C_Lyz from 0 to 8.334 μg/mL (Figure 5B). Since DNA-1 can bind specifically to LYZ, the binding to LYZ weakens the binding to HSA NPs [31,32,33]. These changes decreased the ability of DNA-1 to suppress HSA NPs aggregates. As a result, the remarkable enhancement of the FP value was observed along with the increase of C_Lyz from 0 to 0.417 μg/mL, and the FP value increase slowed down with the increase of C_Lyz from 0.417 to 8.334 μg/mL, as most DNA-1 had been adsorbed on LYZ [32]. The above results demonstrated the feasibility of the FP detection for LYZ based on the HSA NPs/DNA-1 system.

We further evaluated the FP response of the HSA NPs/DNA-1 system to a range of metal ions and biomolecules, including LYZ, Na⁺, K⁺, Ca²⁺, Co²⁺, Cd²⁺, Ba²⁺, Cu²⁺, Zn²⁺, Mg²⁺, Al³⁺,and Cr³⁺, as well as biomolecules such as His, Phe, arginine (Arg), glycine (Gly), L-cysteine (Cys), serine (Ser), Tyr, sarcosine (Sar), Trp, threonine (Thr), trypsin, and pepsin (Figure 6A,B). The data revealed that only the addition of LYZ elicited a significant increase in FP. Moreover, we investigated the FP variations in the HSA NPs/DNA-1 system incubated with 4.167 μg/mL of LYZ in the presence of other metal ions and biomolecules. As shown in Figure 6C,D, despite the coexistence of these substances, LYZ consistently induced a pronounced enhancement in the FP of the HSA NPs/DNA-1 system. The detailed information is summarized in

Table 2. The FP value ratio of HSA NPs/DNA-1 incubated with 4.167 μg/mL of LYZ or various metal ions for 20 min.

Individual Substance	Concentration (μg/mL)	FP Change Ratio (%)
LYZ	4.167	247.0
Na+	4.167	101.6
K+	4.167	124.0
Ca2+	4.167	107.7
Co2+	4.167	115.7
Cd2+	4.167	110.3
Ba2+	4.167	135.0
Cu2+	4.167	112.5
Zn2+	4.167	96.9
Mg2+	4.167	142.2
Al3+	4.167	111.9
Cr3+	4.167	110.1
His	4.167	106.0
Phe	4.167	123.3
Arg	4.167	106.3
Gly	4.167	88.4
Cys	4.167	95.9
Ser	4.167	111.8
Tyr	4.167	104.2
Sar	4.167	148.9
Try	4.167	80.8
Thr	4.167	102.8
Trysin	4.167	150.4
Pepsin	4.167	152.8

and

Table 3. The FP value ratio of HSA NPs/DNA-1 incubated with 4.167 μg/mL of LYZ and various biomolecules for 20 min.

Concomitant Substance	Concentration (μg/mL)	FP Change Ratio (%)
Na+	4.167	249.5
K+	4.167	251.8
Ca2+	4.167	250.0
Co2+	4.167	250.7
Cd2+	4.167	240.4
Ba2+	4.167	258.6
Cu2+	4.167	279.2
Zn2+	4.167	256.7
Mg2+	4.167	228.9
Al3+	4.167	242.9
Cr3+	4.167	245.2
His	4.167	254.0
Phe	4.167	244.9
Arg	4.167	256.9
Gly	4.167	240.6
Cys	4.167	235.4
Ser	4.167	259.8
Tyr	4.167	242.3
Sar	4.167	250.6
Try	4.167	260.1
Thr	4.167	248.7
Trysin	4.167	241.6
Pepsin	4.167	204.5

. In the selective test, only LYZ can trigger a 2.47-fold FP increase (Table 2). In the anti-interference test, the coexistence of those substances can only induce the FP fluctuation of the HSA NPs/DNA-1 and LYZ system in the range from 204.5% to 279.2% (Table 3). These findings demonstrate that the developed HSA NPs/DNA-1 system exhibits selective FP detection capability for LYZ.

Previous studies have demonstrated that aptamers DNA-2 and DNA-3 exhibit high affinity for TBA [34,35]. Based on this observation, we speculated that DNA-2 or DNA-3 preferentially binds to TBA rather than to HSA NPs [36,37]. Figure 7A,B displayed the FP changes in the HSA NPs solution incubated with different concentrations of DNA-2 and DNA-3 for 20 min. The results indicated that the FP value of the HSA NPs solution significantly decreased after the addition of DNA-2 or DNA-3. The varying trend is consistent with the addition of DNA-1. According to the above results, the addition of DNA-1, DNA-2, or DNA-3 can induce changes in HSA NPs from an aggregate state to a dispersed state. The FP value of HSA NPs in a well dispersed state is about 280 mP. Herein, we further investigated the FP changes in the HSA NPs incubated with 2.5 μg/mL of DNA-2 and various TBA concentrations (C_TBA). As shown in Figure 8A, the FP value of the HSA NPs/DNA-2 system gradually enhanced with the increase in C_TBA from 0 to 20.84 μg/mL. Moreover, the relationship between the FP value and C_TBA in the range of 0−20.84 μg/mL can be well described by the following equations:

FP = 303.3 + 20.9 C_TBA

(6)

The fitting coefficient R² was 0.993, and the detection limit for TBA was 0.058 μg/mL according to the 3 s/k criterion. Instead, the fluorescence emission spectra of the HSA NPs/DNA-2 system changed a little with the increase in C_TBA from 0 to 20.84 μg/mL (Figure 8B).

In addition, the FP changes in the HSA NPs incubated with 2.5 μg/mL of DNA-3 and various TBA concentrations are shown in Figure 9A. It is worth noting that the FP value of the HSA NPs/DNA-3 system just exhibited a slight enhancement with the increase in C_TBA from 0 to 20.84 μg/mL. The relationship between the FP value and C_TBA in the range of 0–2.084 μg/mL can be well described by the following equations.

FP = 284.7 + 17.1 C_TBA

(7)

The coefficient R² was calculated to be 0.994, and the detection limit for TBA was determined to be 0.095 μg/mL based on the 3 s/k criterion. Concurrently, the fluorescence emission spectra of the HSA NPs/DNA-3 system exhibited minimal variation with increasing C_TBA ranging from 0 to 20.84 μg/mL (Figure 9B). These findings indicated that the HSA NPs/DNA-2 system demonstrated a more pronounced FP response to TBA compared to the HSA NPs/DNA-3 system. Previous studies have reported that the Kd of aptamer DNA-2 for TBA is 0.5 nM, which is significantly lower than the Kd of aptamer DNA-3 for TBA, reported as 25 nM [38,39]. This suggests that DNA-2 possesses a higher affinity for TBA than DNA-3. The observed FP sensitivity of the HSA NPs combined with the respective DNA sequences to TBA correlates with their respective affinities for TBA. It is therefore hypothesized that the differential FP responses are attributable to variations in binding affinity.

The FP response of the HSA NPs/DNA-2 system to various metal ions and biomolecules was further evaluated (Figure 10A,B). The results indicated that only the addition of TBA elicited a significant increase in FP. Additionally, the FP changes in the HSA NPs/DNA-2 system were examined following incubation with 8.334 μg/mL of TBA in the presence of 8.334 μg/mL of other metal ions and biomolecules. As shown in Figure 10C,D, TBA continued to induce a pronounced enhancement in FP within the HSA NPs/DNA-2 system despite the presence of these coexisting substances. Detailed information is summarized in

Table 4. The FP value ratio of HSA NPs/DNA-2 incubated with 8.334 μg/mL of TBA or various metal ions for 20 min.

Individual Substance	Concentration (μg/mL)	FP Changes Ratio (%)
TBA	8.334	184.0
Na+	8.334	106.5
K+	8.334	112.2
Ca2+	8.334	108.0
Co2+	8.334	101.1
Cd2+	8.334	116.0
Ba2+	8.334	116.1
Cu2+	8.334	95.8
Zn2+	8.334	128.2
Mg2+	8.334	113.3
Al3+	8.334	105.3
Cr3+	8.334	115.8
His	8.334	93.1
Phe	8.334	108.0
Arg	8.334	100.1
Gly	8.334	101.1
Cys	8.334	97.3
Ser	8.334	116.0
Tyr	8.334	106.5
Sar	8.334	101.1
Try	8.334	0.729
Thr	8.334	117.6
Trysin	8.334	135.4
Pepsin	8.334	117.9

and

Table 5. The FP value ratio of HSA NPs/DNA-2 incubated with 8.334 μg/mL of TBA and various biomolecules for 20 min.

Concomitant Substance	Concentration (μg/mL)	FP Changes Ratio (%)
Na+	8.334	183.0
K+	8.334	188.5
Ca2+	8.334	196.7
Co2+	8.334	179.8
Cd2+	8.334	191.9
Ba2+	8.334	184.3
Cu2+	8.334	207.5
Zn2+	8.334	193.7
Mg2+	8.334	177.9
Al3+	8.334	190.0
Cr3+	8.334	193.7
His	8.334	192.5
Phe	8.334	187.9
Arg	8.334	190.8
Gly	8.334	167.6
Cys	8.334	181.7
Ser	8.334	184.1
Tyr	8.334	194.9
Sar	8.334	187.7
Try	8.334	187.3
Thr	8.334	206.2
Trysin	8.334	187.5
Pepsin	8.334	188.8

. In the selectivity assay, only TBA triggered a 1.84-fold increase in FP (Table 4). In the interference-resistance test, the coexistence of these substances resulted in FP fluctuations of the HSA NPs/DNA-2 and TBA system ranging from 167.6% to 207.5% (Table 5). Collectively, these findings demonstrate that the developed HSA NPs/DNA-2 system is suitable for the selective FP-based detection of TBA.

As shown in

Table 6. The detection performance for LYZ and TBA based on various kinds of optical detections.

Analyte	Detection Mode	Detection System	Detection Range (μg/mL)	Detection Limit (μg/mL)	Reference
LYZ	Fluorescence	FAM-labeled aptamer/PFP -SO3Na	0–39.7	0.80	[40]
	Fluorescence	MNP/QD@MIPs	0.286–2.86	0.006	[41]
	Resonance Rayleigh Scattering	GSH−Cd doped ZnSe QDs	0–1.5	0.1	[42]
	FP	FITC-Chito- Oligosaccharid	0.43–3.57	0.43	[43]
	FP	FITC-labeled oligosaccharide	17–87.5	17	[44]
	FP	HSA NPs and DNA	0–0.417	0.032	This work
TBA	Fluorescence	Stilbene/DNA	1.08–3.6	0.738	[45]
	Fluorescence	Recombinant protein/Au NPs	0.0288–28.8	0.0003	[46]
	Fluorescence	DNA-Ag NCs	0.18–3.26	0.03	[47]
	Fluorescence	Ag NCs	0.018–7.2	0.036	[48]
	Fluorescence	Triple-helix aptamer	0.018–4.3	0.006	[49]
	FP	HSA NPs and DNA	0–2.084	0.095	This work

, we summarized the detection performance for LYZ and TBA based on various optical assays. It can be found that this method offered a simple detection system and similar detection range and detection limit [40,41,42,43,44,45,46,47,48,49].

In order to further test the applied potentials of this method in practical samples, the HSA NPs/DNA-1 system and the HSA NPs/DNA-2 system were utilized for the FP detection of LYZ and TBA concentrations in saliva samples. LYZ and TBA were added to 2-fold saliva sample dilutions. The determined results are shown in

Table 7. The detection of LYZ in saliva samples.

	Added (μg/mL)	Tested (μg/mL)	Recovery (%)	RSD (%)
1	2.5	2.3	92	4.2
2	8.0	8.6	108	3.3

and

Table 8. The detection of TBA in saliva samples.

	Added (μg/mL)	Tested (μg/mL)	Recovery (%)	RSD (%)
1	8.0	8.3	104	4.7
2	20.0	21.8	109	3.1

. The added LYZ and TBA were successfully detected, and the recoveries were in the range of 92–108% and 104–109%, with relative standard deviation (RSD) no more than 4.7%. The above results demonstrated the potential of detecting LYZ and TBA in actual samples based on this method.

4. Conclusions

Fluorescent albumin nanoparticles were synthesized, and their FP properties were investigated following incubation with different DNA sequences. The fabricated HSA NPs exhibited a marked reduction in FP upon exposure to DNA-1 and DNA-2, which possessed high binding affinities for LYZ and TBA, respectively. Additionally, HSA NPs/DNA-1 and HSA NPs/DNA-2 complexes were developed, facilitating the FP-based detection of LYZ and TBA via the recovery of FP signals.