1 Introduction

Recently, the good interests in producing and demand of metal oxide nanoparticles in commercial and industrial merchandise have led to target on the potential influence of these particles on biomedicine. In this field, copper-based nanoparticles in an area unit are an issue of nice interest in recent years because of their distinctive physical and chemical properties [1]. Copper oxide (CuO) could be a semiconductor compound with a monoclinic structure. It has fascinated large captivate as a result of having the simplest structure of the family of copper compounds and reveals a variety of potentially helpful physical application like catalysts and biomedical application. Copper oxide (CuO) has attracted attention largely owing their antimicrobial and biocide properties and its other biomedical applications [2, 4]. Few metal oxide nanoparticles, like zinc oxide (ZnO) and copper oxide (CuO), have antimicrobial and antifungal properties that construct them, ideal for a variety of coating applications. Nanostructure CuO is employed in textile fibers, coatings, and thermoplastics to inhibit microbial and fungal growth, additives for ceramics process and colorants, and pigments for different materials [2,3,4]. Copper oxide–based nanofibrillar cellulose may be a promising biomaterial [5]. The unique properties of CuO nanocrystals are significantly interested in the biomedical field increases rapidly. Copper oxide nanoparticles are also used as contrast agents for MRI and ultrasound dual-modality imaging. However, before going into realizing the medical specialty applications, it is greatly essential to grasp the means, nature, and condition of the interaction of clean CuO NPs with protein. However, before going into realizing the biomedical applications, it is very much essential to understand the way, nature, and condition of the interaction of bare CuO NPs with plasma protein. The risks of exposing to nanocrystals for human are sometimes toxic and may be accidental. The most important disadvantage of their use in the medical domain is due to their potentially toxic effects [4, 6,7,8]. CuO NPS can also be toxic to mammalian cells as well as for vertebrates and invertebrates [9, 10]. They also interact with various biomolecules. The CuO NPs directly or indirectly come in contact with the interfaces of different biomolecules including the protein of our body under different biomedical applications. When nanoparticles come in contact with protein molecules, they form a nanoparticle-protein corona and interact with protein molecules. The effect of this CuO NPs on the different biomolecules is different. They interact with different biomolecules including protein molecules due to a large surface free energy in nano-domain biomedicine under contact with our body. The research for the effect of CuO NPs on protein molecules is very few [11,12,13,14]. Nanoparticle-protein interaction depends on various parameters like particle size, shape, the chemical composition of its surface, ζ potential and surface hydrophobicity, pH of the solution, nature of the protein, and temperature [15,16,17,18,19,20]. Also, the concentrations of nanoparticles and the nature of biological fluid can influence the result of identification experiments [21]. Different ranges of concentration-dependent serum protein adsorption at the nano-bio interfaces is studied solely few researchers [22]. Here, we focused the role of CuO NPs concentration due to interaction with BSA protein by spectroscopic and microscopic techniques, mainly focused on the two different rages of concentrations. We studied in details about the long-time dynamics of the energy transfer mechanism under different conditions. We used the bovine serum albumin (BSA) as a model protein because it is inexpensive, easily accessible, and structurally more similar to human serum albumin (HSA).

2 Experimental

2.1 Synthesis of CuO Nanoparticles and Bioconjugate Preparation

In the synthesis of CuO NPs at room temperature, we accompanied the chemical precipitation method for the synthesis of CuO nanostructures. In this technique, 2.5 g copper acetate was dissolved in 50 mL deionized water and stirred in the magnetic stirrer for a few minutes, then 1.00 g NaOH in 50 mL water solution was added drop-wise under a vigorous stirring condition. The pH of the solution is maintained ~ 10. After the complete addition of the NaOH solution within the copper acetated solution, the magnetic stirring was continuing for 2 h. The reaction solution is kept at 35 °C overnight. The product was filtered and washed by di-water several times. The final product power was annealed by a muffle furnace under 300 °C for 2 h. Then the sample is cooled into room temperature and collected for characterization. The BSA solutions with a preset concentration of BSA (CBSA = 0.01 mg/mL) was ready by exploitation triple distilled water, deionized with a Milli-Q water purification system from Millipore, USA. The pH and the resistivity of freshly ready water were 6.8 and 18.2 MΩ cm, severally. The ready CuO NPs were dispersed in deionized Millipore water by ultrasonication for 60 min. The concentration of the CuO was varied from 12.5 to 1257.16 μM. CuO NPs–BSA mixed solutions were prepared by mixing 0.001 mg/mL BSA with CuO, ranging from 12.5μM to 1257.17 μM with proper ratio.

2.2 Characterization Methods

See supplementary S1.

3 Results and Discussion

3.1 Absorption Spectrum

The optical absorption is studied with in wavelength range 200nm to 900 nm. The observed optical absorption spectrum for the variation of optical absorbance of the grown CuO NPs dispersed in water is shown in Fig. 1a. The maximum absorbance peak was seen at ≈ 385.25 nm. The optical absorption coefficient (α) has been calculated from the Lambert law in the wavelength region 200–900 nm [23]. The optical band gap (Eg) of the as-arranged nanoparticles have been figured utilizing the Tauc equation [24]:

$$ {\left(\alpha \mathrm{h}\upnu \right)}^2=c\left(\mathrm{h}\upnu -{E}_{\mathrm{g}}\right) $$
Fig. 1
figure 1

a Absorption spectroscopy of CuO NPs. b Bandgap determination plot. c Plot of lnα versus photon energy (hʋ) of the CuO nanoparticles. d Dynamic light scattering (DLS) spectrum of pure CuO NPs

Full size image

Here, α = 2.303 A/t (A = absorbance). Figure 1b demonstrates the plot of (αhν)2 vs. energy (hν), and it is utilized to decide band gap. The estimated direct band gap is 3.60 eV. This band gap esteem is greater than bulk CuO (3.25 eV) reported by Koffyberg et al. [25]. The greater band gap formation in CuO nanostructure was reported by Klinbumrung et al. [26]. The watched increment in the direct bandgap esteems (a blue move) of CuO with the diminishing in nanoparticle estimate is ascribed to the quantum-confinement. Wavelength reliance of optical absorption property of semiconductor can be communicated by the following condition [27]:

$$ \alpha =\alpha 0\ \exp\ \left[\frac{\mathrm{h}\upgamma}{E_{\mathrm{u}}}\right] $$

By plotting ln(α) with the incident photon energy hγ, the Urbach energy EU can be resolved. The variation of ln(α) with the incident photon energy hγ appeared in Fig. 1c. Urbach energy is acquired from the inverse of the incline (see Fig. 1c). The calculated value of the Urbach energy is 1.026 eV, which is near the announced estimation of 1.01 eV [28]. This minor change from other reported value of Urbach energy may be due to the crystal disorder, temperature, bondings, and change in average photon energies [29].

3.2 Emission Spectrum

Figure 2a and 2b show the photoluminescence spectrum of CuO NPs under two different excitation wavelengths; one is 320 nm and another is 340 nm, respectively. The emission peaks corresponding to excitation (λext) at 320 nm are observed at 426.399 nm, 459.51 nm, and 486.51 nm [30, 31]. The first one corresponds to the band edge emission in the violet region. The second is because of the oxygen vacancies arise in the blue region. The third one arises from the multiple oxidation states of copper in the blue region. The quantum yield of the blue emission peak corresponding to oxygen vacancies is larger compared with other regions of the wavelengths. The quantum yield is directly proportional with the area of the Gaussian fitting at the emission maxima (see supplementary S2 (a)). The emission peaks (λem) corresponding to excitation at 340 nm are observed at 458.75 nm and 487.96 nm [32]. The quantum yield of the blue emission peak at 487.96 nm is larger compared with the peak at 458.75 nm (see supplementary S2 (b)). The corresponding energy levels are tabulated in Tables 1 and 2.

Fig. 2
figure 2

a Emission spectrum of CuO NPs with excitation wavelength 320 nm. b Emission spectrum of CuO NPs with excitation wavelength 340 nm. c XRD spectrum of CuO NPs. d Plot of β1/2cosθ vs. 4sinθ of CuO NPs sample

Full size image
Table 1 Characterization parameters from optical study of CuO NPs
Full size table
Table 2 Characterization parameters from XRD of CuO NPs
Full size table

3.3 Dynamic Light Scattering

The hydrodynamic size of the grown CuO NPs is measured by the DLS technique [33]. All sizes rumored here were supported intensity average. The variation of intensity (%) with size (nm) is shown in Fig. 1d. The light scattering of the CuO nanoparticles (Fig. 1d) dispersed in water showed that the particles are mono dispersed with an average hydrodynamic size of ~ 24 nm.

3.4 XRD

The XRD pattern shown in Fig. 2c shows that the unit cell of the synthesized CuO nano-crystal is monoclinic with the presence of the peaks (110), (\( \overline{1} \)11), (111), (\( \overline{2} \)02), (020), (202),(\( \overline{1} \)13), (022), (220), (\( \overline{3} \)12), and (203) [34, 35]. The nanoparticle size is calculated from the Scherrer formula [36]:

$$ {R}_{\mathrm{hkl}}=\frac{K\leftthreetimes }{\beta\ \mathrm{cos}\uptheta} $$

Here, Rhkl is the crystalline size and K = 0.94, for copper radiation. The average particle size from this equation is found to be ~ 17.88 nm. The strain (ɛ) and grain size (D) of the nanomaterial is also calculated using the Williamson-Hall (W-H) equation [37]:

$$ \beta \cos \theta =\frac{k\uplambda}{D}+4\varepsilon\ \sin \theta $$

The average size (D) and dislocation density d = 1/D2 are calculated [35]. The average crystal size of CuO NPs is found to be 17.88 nm from the Scherrer formula and 22.73 nm from the fitted result of the W-H equation (Fig. 2d). The value of strain is 0.00166 from the fitted result of the W-H equation and 0.0052 from Scherrer equation. The value of dislocation density is 3.127 × 10−3/nm2 from the result of the W-H and 1.928 × 10−3/nm2 from the Scherrer equation. The calculated lattice parameters are a = 4.568 A°, b = 3.4451 A°, and c = 5.211 A° [38].

3.5 Concentration-Dependent Absorption Spectrum of CuO-BSA Bioconjugates

The absorption spectrum of CuO NPs–BSA bioconjugate is shown in Fig. 3. The phenyl group of Tryptophan (Trp) and Tyrosin (Tyr) residues inside BSA molecule produce a maximum absorbance of around ~ 278 nm (shown in Fig. 3a (a)) [39]. The absorbance at 278 nm of the BSA increases as the concentration of the CuO NPs increases within the CuO-BSA bioconjugate. Here, we use the concentration range of CuO NPs from12.5μM to 113.13 μM, noted as small concentration range. The maximum peak height of the free BSA (~ 278 nm) broadens and significantly reduces from its original peak height (Fig. 3a) after the addition of CuO NPs within this range of concentration. The shape of the peak around ~ 278 nm for free BSA also changes after the addition of CuO nanoparticles within the bioconjugate (Fig. 3a). The maximum absorbance peak (at ≈ 385.25 nm) for CuO nanoparticles also increases within the bioconjugate. The total absorbance of the CuO NPs–BSA system increases due to the formation of the ground state complex [40]. We use another set of concentration range from 125.71μM to 1257.16 μM, called large concentration range. The use of the large concentration of the CuO NPs (Fig. 3c) suppressed the protein absorbance peak at 278 nm and CuO NPs absorbance arises significantly. The Trp peak of BSA at 278 nm is suppressed by the large amount of dosages. This may be due to the larger concentration of the CuO NPs which leads to a smaller amount of protein molecules on its surface [41]. The overlay plot of the absorption spectrum of pure CuO NPs, pure BSA, and BSA–CuO NPs bioconjugate is shown in Figure S4 (see supplementary S6).We observe a maximum red shifting of ~ 5 nm and ~ 8 nm for the absorbance of the BSA corresponding the use of the small concentration and large concentration of the CuO NPs. This red shifting of the BSA peak is due to the energy transfer between CuO NPs to BSA molecules. The significant increment within the absorbance intensity at the wavelength of 278 nm is observed. The increment of the CuO nanoparticles concentration leads to spreading of the peptide chains of BSA and advances the exposure of Trp and Tyr residues [42]. The equilibrium for the bioconjugate formation of CuO NPs–BSA complex is given by:

$$ \mathrm{BSA}+\mathrm{CuONP}\overset{K_{app}}{\leftrightarrow}\mathrm{BSA}\dots \mathrm{CuONP} $$
(1)
Fig. 3
figure 3

a Absorption spectra of (a) BSA 0.001 mg/mL, (b) CuO 12.5 μM + BSA, (c) CuO 15.5 μM + BSA, (d) CuO 25.14 μM + BSA, (e) CuO 37.71 μM + BSA, (f) CuO 50.28 μM + BSA, (g) CuO 62.51 μM + BSA, (h) CuO 75.04 μM + BSA, (i) CuO 87.99 μM + BSA, (j) CuO 113.13 μM + BSA. b Plot of 1/(A − A0) vs. 1/Q in the small concentration range. c Absorption spectra of BSA 0.01 mg/mL + (k) CuO 125.71 μM, (l) CuO 251.41 μM, (m) CuO 377.12 μM, (n) CuO 502.81 μM, (o) CuO 628.52 μM, (p) CuO 754.22 μM, (q) CuO 879.91 μM, (r) CuO 1131.32 μM, (s) CuO 1257.16 μM. d Plot of 1/(A − A0) vs. 1/Q in the large concentration range

Full size image

The value of the association constant (Kapp) was calculated from the Benesi and Hildebrand method [43, 44] (see supplementary S3):

$$ \frac{1}{A_{\mathrm{o}\mathrm{bs}\kern0.5em }-{A}_{\mathrm{o}}}=\frac{1}{A_{\mathrm{c}}-{A}_{\mathrm{o}}}+\frac{1}{K_{\mathrm{app}}\left({A}_{\mathrm{c}}-{A}_{\mathrm{o}\kern0.5em }\right)\left[Q\right]} $$
(2)

where [Q] is the concentration of CuO NPs. The variation of 1/(Aobs − A0) versus 1/[Q] corresponding to the use of two different set concentration ranges is shown in Fig. 3b, d. The linear fitting is done by the origin 7 software by using this data. The slop of the linear fitting gives 1/Kapp (AC − A0) and an intercept adequate to 1/(AC − A0) (Fig. 3b, d). The value of Kapp is found to be 6.51 × 103 M−1 and 2.16 × 103 M−1 for the small concentration range and large concentration range, respectively. The three times greater value of the apparent association constant in the low concentration region supporting the large amount of protein on the CuO NPs surfaces leads to a strong protein-CuO NPs complex formation in the ground state than that in the large concentration region.

3.6 Time-Dependent Energy Transfer Between CuO NPs and BSA with Different Dosages of CuO NPs Under Heat Treatment from Steady-State Fluorescence Spectroscopy

The emission intensity of free BSA changes dynamically with respect to time after bioconjugation with CuO nanoparticles (Fig. 5).We studied this dynamic emission changes under heat treatment by tuning the temperature from 288 to 310 K. The observed decrease in the emission intensity corresponding to each temperature is a signature of energy transfer mechanism. The energy transfer efficiency (Eeff) due to dynamic energy changes between CuO NPs and BSA within the CuO NPs–BSA bioconjugate is calculated and analyzed by considering dipole-dipole coupling from the following formula [44, 45]:

$$ {E}_{\mathrm{eff}}=1-\frac{I}{I_0} $$
(3)

Here, I0 is the emission intensity of free BSA and I is the emission intensity of BSA within the BSA–CuO NPs bioconjugate. The free BSA emission at room temperature is shown in Fig. 4a. Figure 5 shows the emission spectra of BSA (C0.001 mg/mL) within the CuO NPs–BSA bioconjugate under different time from associate initial condition of the bioconjugate formation. The variation of room temperature emission and energy transfer efficiency (Eeff) with time of the CuO NPs–BSA bioconjugates (Q[CuO NPs] = 25.14 μM) are shown in Figs. 6a and 7a, respectively. The result shows that the dynamic change within the emission intensity falls rapidly as much as up to 34 h because of the quick rate change of energy transfer efficiency (Eeff); after 34 h, it goes towards a steady state as a result of the steady rate of energy transfer efficiency. Upon binding on the CuO NP surface, the emission intensity of BSA quenched with time, with a slight red shift. The energy transfer occurs due to Trp and Tyr residues present in the BSA structure because BSA is formed of three homologous domains (I, II, III) that contain twenty Tyr residues and two Trp residues [46] (for details, see supplementary S4& S5). The structure of BSA under different views (atoms, bonding, %(insertion code)s, atomic balls) is shown in Fig. 8. The partial diminish in emission of tryptophan (at 340 nm) intensity within the presence of CuO NPs is feasible once the tertiary deformation of adsorbed BSA molecules is altered in such a simplest way that the Trp moieties involve locations within the shut closeness of other fluorescence quenching amino acids [47]. Here, the quenching of the emission intensity of tryptophan attains a steady state after 34 h and ultimately closely saturation arises (spectra from 34–224 h). This indicates that upon preliminary binding on CuO nanoparticle surface, BSA molecules receives tertiary deformation, i.e., a partial loss in its tertiary conformation, and failed to endure forceful conformational changes. At the body temperature, the emission intensity of free BSA falling rapidly up to 24 h (Fig. 6b) in the presence of CuO NP with a quick rate of energy transfer efficiency (Eeff), after 24 h it goes about steady state and rate of energy transfer slows down into saturation value (Fig. 7b). In the case of using the large concentration of the CuO NPs (378 μM), the steady-state (towards) emission of the bioconjugate occurs beyond 45 h and 36 h at room temperature and body temperature, respectively (Fig. 6c and d, respectively). In the small concentration region, the saturated value of Eeff around 45% occurs within 224 h. This value is almost the same as the room temperature and human normal body temperature. The change in Eeff at room temperature is from 23.4 to 45.4%, i.e., ΔEeff = 22%, and at body temperature is from 30.6 to 44%, i.e., ΔEeff = 13.6%. When we use large concentration, a large amount of energy transfer occurs initially due to a large amount of the quencher (CuO NPs), but the rate of energy transfer is slow. Within 224 h, the Eeff changes from 54.4 to 62% at room temperature and that from 54.8 to 61.4% of body temperature. The total change (ΔEeff) is 5.6% at room temperature and 6.6% at body temperature. Hence, the total change in energy transfer within 224 h is almost 6% for both temperatures, i.e., Eeff is almost independent of the temperature for a large concentration of the quencher. In small concentration region, Eeff depends on the temperature, whereas in large concentration regions, Eeff is almost independent of the temperature. Energy transfer strongly depends on the concentration of the CuO NPs used.

Fig. 4
figure 4

Emission spectrum of pure BSA protein at the temperature (a) T = 288 K, (b) T = 303 K, and (c) T = 310 K

Full size image
Fig. 5
figure 5

Emission of BSA–CuO NPs bioconjugate a with BSA = 0.001 mg/mL and CuO NPs = 25.14 μM under different time (0 to 224 h) at room temperature 298 K, b with BSA = 0.001 mg/mL and CuO NPs = 25.14 μM under different time (0 to 224 h) at body temperature 310 K, c with BSA = 0.001 mg/mL and CuO NPs = 378 μM under different time (0 to 224 h) at room temperature 298 K, and d with BSA = 0.001 mg/mL and CuO NPs = 378 μM under different time (0 to 224 h) at body temperature 310 K

Full size image
Fig. 6
figure 6

Variation of emission intensity with time of the bio conjugate. a BSA = 0.001 mg/mL and CuO NPs = 25.14 μM under at room temperature 298 K. b BSA = 0.001 mg/mL and CuO NPs = 25.14 μM at body temperature 310 K. c BSA = 0.001 mg/mL and CuO NPs = 378 μM at room temperature 298 K. d BSA = 0.001 mg/mL and CuO NPs = 378 μM at body temperature 310 K

Full size image
Fig. 7
figure 7

Variation of the energy transfer efficiency (Eeff) with time of the bio conjugate. a BSA = 0.001 mg/mL and CuO NPs = 25.14 μM under at room temperature 298 K. b BSA = 0.001 mg/mL and CuO NPs = 25.14 μM at body temperature 310 K. c BSA = 0.001 mg/mL and CuO NPs = 378 μM at room temperature 298 K. d BSA = 0.001 mg/mL and CuO NPs = 378 μM at body temperature 310 K

Full size image
Fig. 8
figure 8

Structure of BSA. a %(Insertion code)s. b %(Atoms), c %(Atoms)_Sphere. d Bonds

Full size image

3.7 Emission of CuO NPs–BSA Bioconjugate Under Heat Treatment: Fluorescence Quenching Study With Small and Large Concentration Range

We studied the change in the emission spectrum of the pure BSA molecule for the use of small concentration and large concentration range of the CuO NPs under heat treatment (Figs. 9 and 11, respectively). The emission of pure BSA protein at the three different temperatures (288 K, 300 K, 311 K) is shown in Fig. 4. The maximum intensity of free BSA at 340 nm decreases with an increase in temperature due to heat inducing changes within the BSA structure. The maximum emission peak at 340 nm of the BSA due to Trp is modified by the addition of CuO NPs of various concentrations (QCuO = 12.5 to 75.04 μM) under three different temperatures (288 K, 300 K, 311 K). In size modification, the intensity reduces with red shifting in the pure BSA fluorescence spectrum under the addition of different CuO NP concentrations suggesting the prevalence of the fluorescence quenching process (Fig. 9a, b, c) [48]. Red shift of ~ 2 nm is found with QCuO = 75.04 μM and CBSA = 0.001 mg/mL owing to raising hydrophobicity caused by CuO NPs [49]. The binding of CuO NPs with BSA is studied by the documented Stern-Volmer (S-V) equation [50, 51]

$$ \frac{F_0}{F}={K}_{\mathrm{SV}}\left[Q\right]+1 $$
(4)
Fig. 9
figure 9

a Represents the fluorescence spectrum of CuO NPs–BSA bioconjugate at 288 K with CBSA = 0.001 mg/mL + (a) QCuO = 12.5 μM, (b) QCuO = 25.14 μM, (c) QCuO = 37.71 μM, (d) QCuO = 50.28 μM, (e) QCuO = 62.52 μM, (f) QCuO = 75.04 μM. b Fluorescence spectrum of CuO NPs–BSA bioconjugate at 303 K with CBSA = 0.001 mg/mL + (a) QCuO = 12.5 μM, (b) QCuO = 25.14 μM, (c) QCuO = 37.71 μM, (d) QCuO = 50.28 μM, (e) QCuO = 62.52 μM, (f) QCuO = 75.04 μM. c Fluorescence spectrum of CuO NPs–BSA bioconjugate at 310 K with CBSA = 0.001 mg/mL + (a) QCuO = 12.5 μM, (b) QCuO = 25.14 μM, (c) QCuO = 37.71 μM, (d) QCuO = 50.28 μM, (e) QCuO = 62.52 μM, (f) QCuO = 75.04 μM. d Variation of F0/F versus Q plot for CuO NPs–BSA complex at the temperatures (a) 288 K, (b) 303 K, and (c) 310 K in the low-concentration range

Full size image

Here F0 and F are the steady-state emission intensities of BSA within the absence and presence of CuO NPs, respectively, and Q is the concentration of CuO NPs. The values of S-V quenching constant (KSV) for 288 K, 300 K, and 311 K temperatures are summarized in Table 3. Here, we observed the decrement of KSV with increment of temperature from 288 to 300 K which indicates that the quenching mechanism of BSA is a static for the use of low concentration of the CuO NPs, and therefore, the strength of interaction decreases inside this varying temperature [53]. Inside the temperature (T) range 300 to 311 K, we observe the increment of KSV with T signifying that the quenching mechanism of BSA could be a dynamic quenching process in the presence of CuO NPs. Hence, the strength of interaction increases inside these T variations [54]. The observed quenching is a mixed kind, i.e., each dynamic and static quenching is present under a low concentration range of the CuO NPs [40]. The F0/F versus [Q] plots at different temperature are shown in Fig. 9d. (For details of conditions regarding static and dynamic quenching see our previous reports [40, 52].)

Table 3 Binding parameters and thermodynamic parameters of BSA–CuO Nanoparticle interactions. Small-concentration range CuO NPs
Full size table

The emission of BSA–CuO NPs bioconjugate within the large concentration range of the CuO NPs are shown in Fig. 11a, b, c for the temperatures 288 K ,303 K, and 310 K, respectively. The maximum emission peak (340 nm) of the pure BSA is largely modified within the shape and size for such concentration range and the corresponding red shifting is 5 nm. The fluorescence quenching of BSA protein is large under a large concentration range compared with the use of the small concentration range for any particular temperature. Larger protein quenching leads to large protein denaturation with leading energy transfer between a large concentration of CuO NPs and BSA protein. The F0/F vs. [Q] plots at different temperatures within the large concentration range are shown in Fig. 11d.

In the large amount of the dosages of CuO NPs, the value of KSV gently increases with the increase of the temperature from 288 to 311 K (Table 4) signifying that the quenching mechanism is a dynamic nature in the presence of high dosages of CuO NPs and the strength of interaction increases in this varying temperature [55, 56]. The binding constant K and the number of binding sites (n) between BSA and CuO NPs at different temperatures with different range of concentrations are calculated using the below equation [54]:

$$ \log \left[\frac{F_0}{F}-1\right]=\log K+n\ \log \left[Q\right] $$
(5)
Table 4 Binding parameters and thermodynamic parameters of BSA–CuO Nanoparticle interactions. Large-concentration range of CuO NPs
Full size table

The variation of \( \log \Big[\frac{F_0}{F} \) − 1] vs. log[Q] is shown in Figs. 10a and 11a for the use of small concentration range and large concentration range, respectively. The values of n and logK, at 288 K, 300 K, and 311 K, are organized in Tables 3 and 4 for comparison.

Fig. 10
figure 10

a Represents the log[(F0/F) − 1] versus log[Q] plot for CuO NPs–BSA complex at the temperatures (a) 288 K, (b) 303 K, and (c) 310 K in the low-concentration range. b Variation of logK versus 1/T plot for CuO NPs–BSA complex in the low-concentration range

Full size image
Fig. 11
figure 11

a Represents the fluorescence spectrum of CuO NPs–BSA bio conjugate at 288 K with CBSA = 0.001 mg/mL + (a) QCuO = 125.7 μM, (b) QCuO = 377.1 μM, (c) QCuO = 678.5 μM, (d) QCuO = 879.9 μM, (e) QCuO = 1131 μM, (f) QCuO = 1257.16 μM. b Fluorescence spectrum of CuO NPs–BSA bioconjugate at 303 K with CBSA = 0.001 mg/mL + (a) QCuO = 125.7 μM, (b) QCuO = 377.1 μM, (c) QCuO = 678.5 μM, (d) QCuO = 879.9 μM, (e) QCuO = 1131 μM, (f) QCuO = 1257.16 μM. c Fluorescence spectrum of CuO NPs–BSA bioconjugate at 310 K with CBSA = 0.001 mg/mL + (a) QCuO = 125.7 μM, (b) QCuO = 377.1 μM, (c) QCuO = 678.5 μM, (d) QCuO = 879.9 μM, (e) QCuO = 1131 μM, (f) QCuO = 1257.16 μM. d Variation of F0/F versus Q plot for CuO NPs–BSA complex at the temperatures (a) 288 K, (b) 303 K, and (c) 310 K in the large-concentration range

Full size image

The result for the number of binding sites (n) (see Tables 3 and 4) shows that “n” increases with an increase in the temperature range in both use of the lower and higher concentration range of the CuO NPs. In the case of high concentration (Q), the value of n is less than one (n < 1) signifying a negative cooperative reaction between CuO NPs and BSA [57, 58]. However, in the low concentration region, the value of n is greater than one (n > 1) signifying a positive cooperative reaction between CuO NPs and BSA. The large value of the binding sites (n ˃ 1) in all temperatures of the lower concentration region is due to the large amount of protein molecules in the surface supporting the absorption spectrum of the CuO-BSA bioconjugate [57, 58]. The value of n is small (< 1) in the high Q range corresponding to smaller amount of BSA molecules on its surface. The thermodynamic parameters (change in enthalpy, ∆H; change in Gibb’s free energy, ΔG; change in entropy, ∆S) for BSA and CuO NPs bioconjugates are calculated using the van ’t Hoff equation [59]

$$ \ln K=-\frac{\Delta H}{RT}+\frac{\Delta S}{R} $$
(6)

The variation of logK vs. 1/T graph for CuO NPs–BSA bioconjugate is shown in Figs. 10b and 12b for the use of small concentration and large concentration range, respectively. The Gibbs free energy change is:

$$ \Delta G=\Delta H-T\Delta S=- RT\ln K $$
(7)
Fig. 12
figure 12

a Represents the log[(F0/F) − 1] versus log[Q] plot for CuO NPs–BSA complex at the temperatures (a) 288 K, (b) 303 K, and (c) 310 K in the large-concentration range. b Variation of logK versus 1/T plot for CuO NPs–BSA complex in the large-concentration range

Full size image

The thermodynamic parameters of the CuO NPs–BSA bioconjugate are summarized in Tables 3 and 4 [59]. We find that in ΔG, ΔH is negative and ΔS is positive. Hence, spontaneous binding occurs in an exothermic interactive way [60,61,62]. The positive ∆S constitutes that electrostatic interaction is the leading force for binding the BSA on CuO NPs [63, 64]. Electrostatic binding between BSA and oxide materials were studied by Sun et al. [65]. Also, the hydrophobic interaction is present along with electrostatic interaction. The hydrophobic interaction is confirmed from red shift of the fluorescence peak of free BSA in the presence of CuO NPs. The interactive forces involved in the BSA–CuO NPs conjugate is independent of the concentration of CuO NPs used.

3.8 Time-Resolved Fluorescence on BSA–CuO NP Bioconjugate and Energy Transfer Within the Conjugate

Time-resolved fluorescence activity is a vital and sensitive technique to understand the result of the microenvironment encompassing a fluorescent molecule [66, 67]. To determine the origin and efficiency of the energy transfer between BSA and CuO NPs, excited-state lifetimes of BSA and BSA–CuO NPs bioconjugate were measured by time-resolved fluorescence spectroscopy. Measurements were performed for free BSA and so upon the addition of CuO NPs to the BSA solution. We have measured the fluorescence lifetime of BSA protein within the CuO NPs–BSA bioconjugate (Fig. 13). The decay curve of free BSA was fitted bi-exponential with lifetimes of 3.26 ns and 6.61 ns (see Table 5). Recent studies have shown that the shorter lifetime is freelance of any structure and is characterized as an interior property and organization of the tryptophan structure within the excited state [68]. We consider the average value of lifetime instead of individual component. The calculated average lifetime is 4.94 ns for free BSA molecule. The fluorescence lifetime of BSA was found to have decreased in the presence of CuO nanoparticles (see Fig. 13) as shown in Table 5. This was presumably due to energy transfer from the adsorbed protein to the CuO nanoparticle. The lifetime results clearly concluded that the quenching of fluorescence in the presence of CuO NPs is dynamic in nature.

Fig. 13
figure 13

The kinetic of the fluorescence decays (TCSPA) of (a) pure BSA (0.001 mg/mL) and (b) BSA + CuO NPs bioconjugate

Full size image
Table 5 Lifetime from time-resolved fluorescence measurement
Full size table

The energy transfer efficiency (φET), that is that the fraction of photons absorbed by the donor that are transferred to acceptor, will be determined from the PL lifetimes according to the bellow equation [69, 70]:

$$ {{}^{\varphi}}_{\mathrm{ET}}=1-{\tau}_2/{\tau}_1 $$

where τ1 and τ2 are the lifetime of the free BSA protein and adsorbed BSA protein within BSA–CuO NP bioconjugates, respectively. The calculated energy transfer efficiency is 79.35% for the bioconjugate. The calculated φET is greater than 70% (φET > 70%) which confirms that the Förster resonance energy transfer (FRET) mechanism is the key of energy transfer mechanism within the bioconjugate. A schematic representation of FRET mechanism between BSA and CuO NPs is shown in Fig. 16a.

3.9 Transmission Electron Microscopy and Dynamic Light Scattering of the CuO NPs–BSA Bioconjugate

TEM or HRTEM images in Fig. 14 clearly show that nanoparticles are surrounded by protein molecules by the formation of corona in the bioconjugate. Here, BSA molecules formed corona on the surface of CuO NPs within the bioconjugate. Figure 14 shows the direct confirmation of the formation of BSA–CuO NPs corona. The clear image of the individual corona clearly observes form the TEM images for small concentration of the CuO NPs. The outer layer of BSA is bound to the formation of the corona. The magnified image of corona represents that almost spherical CuO NPs with diameter 20 nm are strongly attached with BSA (Fig. 14b). Inset of Fig. 14b shows the zoom part of the image of a corona. This image clearly represents that the core CuO NPs are fully surrounded with BSA along with a shell thickness of ~ 8 nm. This shell thickness of the BSA layer is equal with the dimension of BSA monomer (8 nm) [71]. The individual corona formation is investigated from DLS measurements. The average hydrodynamic size of the protein corona in DLS for the dosages of 12.5 μM CuO NPs is ~ 12 nm, which is slightly greater than BSA monomer (Fig. 15). This slight greater value of the corona arises due to hydrodynamic size. Hence, DLS also supports the TEM results of the corona size. The use for large concentration of CuO NPs destroyed the single corona formation mechanism. Aggregation of the CuO NPs inside the BSA molecule matrix occurs for the use of the large concentration of CuO NPs within the CuO NPs–BSA bioconjugate. This aggregation was clearly observed from the TEM images. The spherical CuO NPs are attached one by one and formed a chain-like structure inside the BSA molecules matrix (Fig. 14d). Hence, selectively single corona formation for large doses is destroyed and the tendency of the aggregation of the BSA molecules and CuO NPs is prominent. As the concentration of the CuO NPs increases, the tendency of clustering increases, and hence, the hydrodynamic radius of the CuO NPs–BSA bioconjugates increases (see Fig. 15a (b), (c), (d)). Adding 0.001 mg/mL BSA at room temperature with 12.5 μM CuO nanoparticles (Fig. 15a (a)), the particle size distribution (PSD) of the CuO NPs–BSA bioconjugate shows the near-monodispersed nature with small broadening. This broadening is large for the use of 50.28 μM CuO NPs (Fig. 15a (b)). This broadening is mainly due to corona formation within the bioconjugate around the single CuO nanoparticle. Hence, a small concentration dose is responsible for selective corona formation. The average hydrodynamic diameter (Dh) of the bioconjugate is 33 nm and 49 nm for the use of 12.5 μM and 50.28 μM doses, respectively. The Dh value and broadening of the PSD increases from 49 to 80 nm with an increasing use of 377.1 μM doses (Fig. 15a). This indicates the formation of few protein aggregations leads to the tendency of nanoparticle agglomeration. Two extra peaks and large broadening of the PSD for the use of 879.9 μM CuO NPs show that the addition of BSA with this concentration of CuO NPs causes the formation large BSA aggregation around single CuO NPs, and the tendency of nanoparticle agglomeration is large. Schematic diagram of CuO nanoparticle exposure concentration effect on BSA protein adsorption is shown in Fig. 16b.

Fig. 14
figure 14

TEM image of the CuO NPs–BSA bioconjugate. a, b BSA = 0.001 m/mL + CuO NP = 24.14 μM. c, d BSA = 0.001 m/mL + CuO NP = 378 μM

Full size image
Fig. 15
figure 15

a DLS spectra of CuO NPs–BSA bioconjugate with (a) BSA = 0.001 mg/mL + CuO NPs = 12.5 μM, (b) BSA = 0.001 mg/mL + CuO NPs = 50.28 μM, (c) BSA = 0.001 mg/mL + CuO NPs = 377.1 μM, and (d) BSA = 0.001 mg/mL + CuO NPs = 879.9 μM. b Variation of the corona size with concentration of CuO NPs

Full size image
Fig. 16
figure 16

a Schematic diagram of the energy transfer mechanism between CuO NPs–BSA bioconjugate. b Schematic representation of corona formation between protein and different-concentration CuO nanoparticles: (a) small concentration of CuO NPs, (b) medium concentration of the CuO NPs, (c) large concentration of the CuO NPs

Full size image

4 Conclusion

We successfully grown the CuO NPs by the cost-effective chemical precipitation method and studied their optical and structural properties. Our CuO NPs are quantum mechanically confined for a large band gap (3.60 eV) with average size 20.31 nm and 1.026eV Urbach energy. The photoluminescence spectrum of pure CuO NPs showed the formation of oxygen vacancies and multiple oxidation states of copper with strong shallow-deep-level blue emission. The crystal unit cell of the nanoparticles is found to be monoclinic with an average size of 20.31 nm and the following lattice parameters: a = 4.568Ao, b = 3.4451A0, c = 5.211A0. Our CuO nanoparticles can be utilized in semiconductor-based optoelectronic devices and in biophotonics. The observed red shifting is 3 nm and 6 nm for the absorbance of the BSA corresponding to the use of the small concentration range and large concentration range of the CuO NPs, respectively. We observe that the lower concentration of the CuO NPs leads to a large amount of protein molecules for each particle surface, whereas a large concentration of the CuO NPs leads to a small amount of protein molecules for each particle surface with the apparent association constant (Kapp) 6.51 × 103 M−1 and 2.16 × 103 M−1, respectively. In a small concentration region, energy transfer efficiency (Eeff) depends on the temperature, whereas in large concentration regions, Eeff is almost independent of the temperature. Energy transfer strongly depends on the concentration of the CuO NPs used. The total changes in energy transfer efficiency (ΔEeff) at room temperature are 22% and 5.6% for the use large and small concentrations, respectively; at body temperature, these changes are 13.6% and 6.6% for the large and small concentrations of the doses, respectively. The quenching in the fluorescence emission of tryptophan (Try) residues in the structure of BSA in the presence of CuO NPs is static in lower temperature and dynamic in higher temperature in the low-concentration range and fully dynamic in the high-concentration range. The concentration of CuO NPs affected the nature of protein denaturation process. The interaction between Try of BSA and CuO NPs is exothermic, electrostatic, and hydrophobic in nature in all concentrations of CuO NPs. Time-resolved fluorescence spectroscopy showed 79.35% energy transfer between BSA and CuO NPs which corresponds to FRET mechanism. The use of small concentration of CuO NPs represents that the core CuO NPs are fully covered by BSA with shell thickness of ~ 8 nm. The use of large concentration leads to aggregation rather than individual corona formation. This study provides important insight into the interaction of CuO nanoparticles with proteins, which may be of importance for further application of these nanoparticles in biomedical applications. The CuO NP–based energy transfer can be used effectively for biomolecular recognition and FRET-based biosensors. This report may be the important experimental report about the role of the nanoparticles concentration within the nano-bio interface science.