Determination of the Influence of Various Factors on the Character of Surface Functionalization of Copper(I) and Copper(II) Oxide Nanosensors with Phenylboronic Acid Derivatives

In this work, we attempt to determine the influence of the oxidation state of copper [Cu(I) vs Cu(II)], the nature of the interface (solid/aqueous vs solid/air), the incubation time, and the structure of N-substituted phenylboronic acids (PBAs) functionalizing the surface of copper oxide nanostructures (NSs) on the mode of adsorption. For this purpose, 4-[(N-anilino)(phosphono)-S-methyl]phenylboronic acid (1-PBA) and its two analogues (2-PBA and bis{1-PBA}) and the copper oxide NSs were synthesized in a surfactant-/ion-free solution via a synthetic route that allows controlling the size and morphology of NSs. The NSs were characterized by scanning electron microscopy, ultraviolet–visible spectroscopy, Raman spectroscopy, and X-ray diffraction, which confirmed the formation of spherical Cu2O nanoparticles (Cu2ONPs) with a size of 1.5 μm to 600 nm crystallized in a cubic cuprite structure and leaf-like CuO nanostructures (CuONSs) with dimensions of 80–180 nm in width and 400–700 nm in length and crystallized in a monoclinic structure. PBA analogues were deposited on the surface of the copper oxide NSs, and adsorption was investigated using surface-enhanced Raman spectroscopy (SERS). The changes in the orientation of the molecule relative to the substrate surface caused by the abovementioned factors were described, and the signal enhancement on the copper oxide NSs was determined. This is the first study using vibrational spectroscopy for these compounds.


INTRODUCTION
Arylboronic acids are a class of chemical compounds commonly used in modern synthesis to form C−C and Cheteroatom bonds. 1 These acids exhibit a reversible coordination profile that is used as a tool for the construction of stimulus-dependent biconjugates used in pharmaceuticals (e.g., antibiotics 2 ), polymers, 3 organic synthesis, electrochemistry, catalysis, 4 materials chemistry (e.g., to obtain predictably organized crystalline materials 5 ), or separation processes. 6,7 Derivatives of phenylboronic acid (PBA) are used in medicine, for example, in selective drug delivery, 8 live cell imaging, 9 cancer treatment (e.g., in the boron neutron capture therapy 10 and for efficient tumor-targeted chemotherapy with doxorubicin−PBA nanocomplexes 11 and low-molecular-weight gels based on PBA derivatives 12 ), in enzyme and HIV inhibition. 10,13 PBA are also used in the development of new fluorophores and chemical sensors for glucose in blood 14,15 or other body fluids. 16 The use of PBA in the treatment of diabetes is based on their specific binding to 1,2-diols or polyols and the formation of reversible covalent PBA/diol complexes. 17 The formation of boronic acid esters is favored near or above the pK a of boronic acid. In order to modify the pK a of PBA and their efficiency in ester formation, many attempts have been made to synthesize various substituted PBA derivatives. 18 For example, it was found that the addition of electron-withdrawing groups to the aromatic ring can lower the pK a by inductive effects, while the addition of electron-donating substituents can increase the pK a . 18 Wulff et al. found that the addition of a nitrogen atom can facilitate the formation of boronate esters. 19 On the other hand, the placement of the carbonyl group facilitates the formation of the boronate ester over almost the entire pH range due to the interaction between boron and carbonyl oxygen. 20−22 However, compounds containing a carbonyl group have a two-dimensional structure, and the lone pair of electrons of the atoms adjacent to the carbonyl group interacts quite strongly with this group. A similar conjugation is not as pronounced in phosphates with a tetrahedral configuration. Therefore, (amino)phosphonic groups are increasingly used in place of carbonyl groups in the rapidly developing field of biochemistry; 23−25 although, much work remains to be done in this area. For the abovementioned reasons, we have synthesized N-substituted 4-[(NH-R)(phosphono)-S-methyl]phenylboronic acids for our research.
The development of metallic nanostructures (NSs), such as Ag, Au, and Zn nanoparticles (NPs) and semiconductor quantum dots surface modified with PBA derivatives also contributed to the exploration of the use of the NSs for the dynamic quantification of glucose in a physiologically important concentration range of 0−20 mM and pH 7.4, 26,27 for the self-regulatory delivery of insulin at a physiological pH, 28 and for the detection of sialic acid as a diagnostic and therapeutic agent in cancer. 29−33 Despite many studies, both the description of the adsorption mode of PBA derivatives and the change of adsorption under the influence of different environmental conditions and the use of copper NPs have been rather neglected, although Cu has a greater biological significance than Ag or Au. 34,35 Accurate adsorption characteristics of PBA derivatives on the surface of NSs is crucial because changes in the intensity of PBA modes can be misinterpreted. That is, changes in the intensity of PBA signals are interpreted quantitatively (e.g., low-intensity signallow compound concentration, high-intensity signalhigh compound concentration) without taking into account the fact that the intensity changes can be associated with a change in the orientation of PBA on the metal surface. Such errors may lead to decreased medical relevance of surface-modified NPs with PBA.
The importance of PBA, the enhanced catalytic activity of copper oxides (as Cu is rapidly oxidized under physiological conditions) in the destruction of cancer cells 36−38 and the advantages of surface-enhanced Raman spectroscopy (SERS)a technique used in a variety of fields, 39−49 which allows us to describe the behavior of a selected molecule at the solid/liquid and solid/air interfacesled us to study the adsorption of N-substituted 4-[(NH-R)(phosphono)-Smethyl]phenylboronic acid and the changes in adsorption due to changes in the chemical structure of the substituent R− (see Figure 1), oxidation state of Cu (copper(I) (Cu 2 O) vs copper(II) (CuO)), and interface type (solid/liquid vs solid/ air).  (Table 1) were synthesized according to the procedure described previously. 50 The purity and chemical structure of the compounds were verified by 1 H, 13 C, 31 P, and 11 B NMR (Bruker Avance DRX 300 MHz spectrometer, Bruker Polska, Poznan) and ESI-MS spectroscopy (Bruker MicrOTOF-Q spectrometer, Bruker Polska, Poznan).

MATERIALS AND METHODS
2.2. Synthesis of Colloidal Cu 2 ONPs and CuONSs. Copper(I) oxide (cuprous oxide, Cu 2 ONPs) and copper(II) oxide (cupric oxide, CuONSs) nanostructures (NSs) were prepared by chronoamperometry (at room temperature using a VoltaLab potentiostat PGZ301 and at a constant electrode potential of 0.8 V for 4 h). 51,52 0.1 M aqueous solution of lithium chloride (LiCl; from Sigma-Aldrich) was freshly prepared and used for CuONS synthesis, while an ethanolic LiCl solution with 10% water was freshly prepared and used for the synthesis of Cu 2 ONPs. The electrochemical treatment was carried out under an inert atmosphere by slowly bubbling the solution with argon gas in a conventional three-electrode cell with a platinum wire as a counter electrode and an Ag/AgCl (1 M KCl) electrode as a reference electrode (the potential is indicated against this electrode). A copper rod served as the working electrode. Before electrochemical treatment, metallic copper (99.99% Cu) was polished with sandpaper to reduce the grain size and then purified in anhydrous ethanol (99.8%; from Sigma-Aldrich). The precipitated product was in the form of orange Cu 2 ONPs and brown CuONSs.
2.3. Ultraviolet−Visible Spectrum Measurements. The ultraviolet−visible spectra (UV−vis) spectra of an aqueous sol and a sample/sol system, measured after 180 min of mixing, were recorded using a LAMBDA 25 UV−vis spectrometer.
2.4. Scanning Electron Microscopy Measurements. The scanning electron microscopy (SEM) images of an aqueous sol were acquired using a SEM instrument, model S-5000 (Hitachi Ltd., Japan), operated at 20 kV.
2.6. Raman and SER Measurements. Aqueous solutions of the studied compounds were prepared by dissolving each compound in deionized water (18 MΩ·cm −1 ; sample concentration 10 −4 M). 10 μL of the sample solution was mixed with 20 μL of aqueous sol solution. The 20 μL of the sample/sol mixture was applied to a glass plate, and the SERS spectra were recorded (no measurements were made for the dried droplet). The spectra were recorded three times at three different locations on each surface.
The Raman and SERS spectra were recorded using a HoloSpec f/ 1.8i spectrograph (Kaiser Optical Systems Inc.) equipped with a liquid-nitrogen-cooled CCD detector (Princeton Instruments). The 785.0 nm line of a NIR diode laser (Invictus) was used as the excitation source. The laser power at the sample position was set to ∼15 mW. The typical exposure time for each SERS measurement was 40 s with four accumulations. The spectral resolution was set to 4 cm −1 . The SERS spectra of a given adsorbate on a given substrate were almost identical, except for small differences (up to 5%) in some band intensities. No spectral changes that could be associated with the decomposition of the sample were observed in these measurements.
2.7. Spectral Analysis. Spectral analysis was performed using a GRAMS/AI program (Galactic Industries Co., Salem, NH).
Several unseparated bands were fitted using the GRAMS/AI program (Galactic Industries Co., Salem, NH). A 50/50% Lorentzian/Gaussian band shape was assumed and fixed for all bands.   53 The 2θ values and [(hkl)] planes in Figure Figure 2D shows a Raman spectrum of Cu 2 ONPs. Characteristic Raman bands of Cu 2 O are observed at 148 (T 1u symmetry), 182, 219 (strongest E u ), 420, 486, and 630 cm −1 (T 1u ) and are in agreement with data from literature. 56−58 The 148 and 219 cm −1 spectral features are due to rotations of the Cu tetrahedron around its center. The 630 cm −1 band is attributed to an out-of-plane vibration of the Cu and O sub-lattice and, like the 148 cm −1 band, is activated by defects. Figure 3 at different magnifications (A scale bar 2 μm and Bscale bar 300 nm). These images show that the monodisperse CuONSs have a leaf-like structure with average dimensions of 80−180 nm in width and 400−750 nm in length. Moreover, image B shows that these structures are composed of small spherical particles that are self-aligned, which is consistent with previous data on the directional growth of CuO nanocrystals along the axis. 59−61 Monodisperse CuONSs form a honeycomb-like skeleton consisting of interconnected networks of sub-micrometer pores 2−3 μm in diameter and 1−1.5 μm thick ( Figure 3A).

Properties of CuONSs. SEM images of CuONSs obtained by the anodic dissolution of Cu are shown in
A UV−vis spectrum of the bare leaf-like CuONSs (black dashed line) and that of the sample adsorbed on their surface (sample/CuONSs; black solid line) are also shown in Figure 3. No optical absorption band at ∼290 nm is seen in the UV−vis spectrum of the bare, large-area, self-assembled CuONSs. 6,62 However, a weak absorption at 219 nm is observed in this spectrum ( Figure 3C, black dashed line), which is attributed to  The Raman spectrum of CuONSs in Figure 3D shows the formation of the pure monoclinic CuO structure (space group C2/c). 59, 69 For the monoclinic structure with two CuO molecules in the unit cell, the group theory predicts six IR-  Langmuir pubs.acs.org/Langmuir Article active (of 3A u + 3B u symmetry) and three Raman-active (A g + 2B g ; of oxygen vibrations) optical modes. 70 The three Ramanactive modes are observed at 295 (A g ), 340 (B g ), and 604 cm −1 (B g ) ( Figure 3D). The XRD pattern used to characterize the size and size distribution of the crystalline CuO domains is shown in Figure  3E. The diffraction peaks at 2θ = 32. 29 71 The pronounced intensity of the diffraction peaks indicates the highly crystalline nature of the CuONSs. The assignment of bands in these spectra (mainly based on the density functional calculation for PBA derivatives 72 ) is given in Table 1.
As can be seen in Figure 4B, the SERS spectrum of 1-PBA adsorbed from aqueous solution on the surface of CuONSs is dominated by two broad bands at 584 (full width at half maximum, fwhm = 58 cm −1 ) and 670 cm −1 (fwhm = 33 cm −1 ) (see Table 1 for the assignment of these bands). These bands are not visible in the corresponding Raman spectrum ( Figure  4A). Therefore, it can be assumed that the 1-PBA molecule binds to the CuONSs surface via the fragment −C(N)PO−. This implies that the free electron pair on the oxygen (of the phosphonic acid group) and the nitrogen atoms are in direct contact with the substrate surface. Considering the sp 2 and sp 3   With the change of the substrate from CuONSs to Cu 2 ONPs, the bands originating from the vibrations of the PO fragment lose intensity or disappear (at 1286 and 670 cm −1 ) ( Figure 4C). The disappearance of the band at 584 cm −1 (on CuONSs) and the increase of intensity at 524, ∼600, and 1383 cm −1 in Figure 4C (on Cu 2 ONPs) are also observed. On the other hand, the wavenumber of the SERS signal at 789 cm −1 [ν(B−C) + ν(B−O)] shifts to 781 cm −1 , and the intensity increases. To explain these observations, it can be assumed that the phosphonic acid fragment is away from the Cu 2 ONP surface and the sp 3 orbital of the boron oxygen atom occupied by the free electron pair has a vertical orientation with respect to this surface. In this orientation, the Ph B(OH) 2 ring is tilted by about 70°with respect to the surface normal, that is, it adopts a nearly horizontal orientation on the surface of the substrate, while the Ph ring adopts a nearly vertical orientation.
In the case of 2-PBA adsorbed on CuONSs and Cu 2 ONPs, the change in the oxidation state of copper also leads to changes in the adsorption mode. For this molecule immobilized on Cu 2 ONPs ( Figure 5C), a strong enhancement of the bands attributed to the vibrations of the aromatic ring (in particular, the 1005 cm −1 band), together with the absence of the wavenumber shift and band broadening compared to the SERS spectrum on CuONSs, is evidence of the vertical arrangement of the aromatic ring on the Cu 2 ONP surface. Also, the absence of spectral features at 937, 789, and 551 cm −1 for 2-PBA on Cu 2 ONPs ( Figure 5C), which are the most intense bands in the SERS spectrum of this molecule on CuONSs ( Figure 5A), indicates the absence of C−N··· Cu 2 ONP, P−O···Cu 2 ONP, and B(OH) 2 ···Cu 2 ONP interactions.
The SERS spectrum recorded immediately after the addition of bis{1-PBA} to the CuONSs sol ( Figure 6B) is dominated by the Ph B(OH) 2 ring modes (ν 18a, ν 19b , ν 9a , ν 18a , ν 12 , ν 1, ν 6b , and ν 16b (see Table 1)), 73 of which ν 12 is the most intense. Again, the changes in the intensity (Raman vs SERS) of these bands indicate the presence of the ring in the perpendicular orientation on the CuONS surface. Moreover, the absence of the characteristic δ(BOH) mode (at about 1075 cm −1 ) indicates that the −B(OH) 2 group is not involved in the interaction with the substrate, so that the 786 and 523 cm −1 bands are due to ring vibrations. When the substrate is changed from CuONSs ( Figure 6B) to Cu 2 ONPs ( Figure 6C), there is a strong enhancement of the band at 1045 cm −1 accompanied by SERS signals of the intermediate intensity at 1015, 881, 797, and 672 cm −1 (see Table 1 for band assignments). These bands indicate the adsorption of bis{1-PBA} by the Ph B(OH) 2 ring, which is arranged more or less  For the further modification of the molecular structure by doubling the fragment of 4-[(N-anilino)(phosphono)-Smethyl]phenylboronic acid (bis{1-PBA}), a closer contact between the molecule and the CuONS surface can be proposed, maintaining the orientation of the Ph B(OH) 2 ring and limiting the contact between the −C(N)PO− fragment and the CuONSs. This conclusion is based on the intense Ph B(OH)2 modes (see the Results and Discussion section above) and a slight broadening of the bandwidth for these modes.
In the SERS spectra of the studied analogues adsorbed on the Cu 2 ONP surface, the following changes were observed under the influence of the structural modifications. 3.6. Influence of Interface Type on Surface Functionalization. The change of the interface from Cu 2 ONPs/water ( Figures 4C, 5C, and 6C) to Cu 2 ONPs/air (Figures 4D, 5D, and 6D) leads to a change of the spectral profile, that is, the adsorbed geometry. Briefly, for 1-PBA at the Cu 2 ONP/water interface, the very strong 781 cm −1 SERS signal is significantly broadened (Δ fwhm = 50 cm −1 ) and has an asymmetric shape.
The decomposition of this band shows that it contains two principal components at 781 and 760 cm −1 [δ(ring)]. The very intense spectral features at 1003 and 1038 cm −1 decrease significantly in intensity compared to those for 1-PBA at the Cu 2 ONP/water interface, while the SERS signals at 992 c and 1029 c cm −1 disappear. This indicates that only one of the rings is in contact with the Cu 2 ONP/air interface in a tilted orientation. Because the bands at 781 and 1075 cm −1 are enhanced in the spectrum, it can be assumed that this ring is a Ph B(OH) 2 ring.
2-PBA is adsorbed at the Cu 2 ONP/water interface by the vertical Ph ring (see the Results and Discussion section above) ( Figure 5C), whereas the ring at the Cu 2 ONP/air interface is not in contact with the substrate surface, as indicated by a slight enhancement of the 1005 cm −1 band, which does not shift in the wavenumber and increases in the bandwidth. The fragment −C(N)PO− is responsible for the adsorption of 2-PBA at the Cu 2 ONP/air interface, as confirmed by the pronounced 937 cm −1 SERS signal and the broad band with maxima at 632, 622, and 583 cm −1 (see Table 1 for band assignments).
In the case of bis{1-PBA} at the Cu 2 ONP/air interface, the intensity of a 1045 cm −1 band decreases and is therefore masked by a 1032 cm −1 band whose intensity increases as does the intensity of the SERS signal at 1005 cm −1 . At the same time, the SERS signals at 881 and 797 cm −1 are attenuated and enhanced, respectively, and the spectral feature at 523 cm −1 disappears. Considering the assignment of the above bands to the modes proposed in Table 1, we can conclude that the change of the interface brings the Ph B(OH) 2 ring closer to the surface, with simultaneous positioning perpendicular to the Langmuir pubs.acs.org/Langmuir Article Cu 2 ONP/air interface. The average intensity of the 797 cm −1 band also suggests that the B−O has an angular orientation with respect to this interface. For this to be possible, the Ph B(OH) 2 ring must be in contact with the substrate via the C 2 − C 3 atoms of the ring. 3.7. Mechanism of Enhancement. The enhancement factor (EF) quantitatively evaluates the effectiveness of the SERS substrate. The most commonly used definition of EF is EF = (I SERS /c SERS )/(I RS /c RS ), where I SERS and I RS are the Raman intensities of SERS and non-SERS substrates, respectively, while c SERS and c RS are the analyte concentrations used for SERS and non-SERS substrates, respectively. 75 For the same analyte concentrations, EF is equal I SERS /I RS . The calculated EF is up to 10 6 orders of magnitude for Ag and Au@ SiO 2 , 10 5 orders of magnitude for Au, 10 4 orders of magnitude for Cu and Ti; 10 3 orders of magnitude for ZnO, CuO, Cu 2 O, TiO 2 , and γ-Fe 2 O 3 ; and 10 2 orders of magnitude for Zn and Fe. 76,77 The mechanism of the enhancement can be predicted from the SERS spectra. When the adsorbate is physisorbed on the metal surface [electromagnetic (EM) mechanism], its SERS spectrum resembles the Raman spectrum of the free molecule. 78,79 When the adsorbate is chemisorbed on the metal surface (charge transfer (CT) mechanism), the formed adsorbate−molecule complex leads to drastic changes in the wavenumbers and intensities of the SERS bands of the adsorbate compared to the corresponding Raman bands. 78 The results of Otero and colleagues have shown that the CT mechanism is responsible for the enormous SERS intensity of the ν 8a mode, which can be used as a marker band to detect and estimate the enhancement produced by the CT mechanism for an adsorbate with an aromatic ring (e.g., benzene, pyridine, pyridazine, and derivatives). 80−82 Considering the above information and the fact that (1) the intensity, width, and wavenumber of the adsorbate bands are only slightly changed compared to these values in the Raman spectrum and (2) there is no particular enhancement of the band due to the ν 8a mode, it can be concluded that on the tested substrates the EM mechanism is responsible for the signal enhancement.

CONCLUSIONS
Compared to their precursor, boronic acid, PBA derivatives show a stronger and more selective antimigratory response to cancer cells in the short term while decreasing the long-term viability of these cells. 83,84 These properties make PBA analogues promising compounds for new cancer therapies. This motivates us to search for and develop new PBA analogues that can selectively inhibit the metastatic properties of various cancer cells.
On the other hand, it has been shown that the unique property of PBA analogues is that they can reversibly bind diols in a covalent manner, which allows, for example, the measurement of glucose fluctuations or the recognition of sialic acid (the expression of sialoglycans in neoplastic cells is observed 85 ), giving them great potential for therapeutic diagnostics. 86−90 Non-enzymatic glucose sensors based on copper or copper oxide/hydroxide NPs have also been developed. 91−97 The presence of copper has been shown to increase the rate of the glucose oxidation reaction and the stability of the sensor itself. It has also been shown that boronic acid groups immobilized on the surface of copper oxide NPs can form reversible covalent bonds with diol groups of glycoproteins on the surface of the microbial cell, which greatly increases the antimicrobial or antifungal activity of these NPs. 98,99 However, it should be kept in mind that copper oxide NPs need to be strictly regulated due to the toxic effect of Cu(II) ions released into the body, which can cause neurodegenerative diseases. 100 Therefore, the prepared sensors containing boric acid can be used to detect copper ions. 101,102 Therefore, not only are PBA analogues being sought after but techniques are also being developed to enable the detection of these molecules, such as the highly sensitive and selective SERS technique. However, the vast majority of these studies focus mainly on the detection capabilities of SERS and are concerned only with the structure of boronic acid derivatives and the nature of the molecular recognition process, ignoring the immobilization of PBA molecules on metal surfaces. Insights into the behavior of immobilized molecules and the intermolecular interactions between the functional groups during molecular recognition are therefore highly desirable for the proper design of SERS sensors. In the absence of such detailed studies, we have performed and described them in this work for a newly developed potential biosensor combining the properties of copper oxide NPs and PBA.
In Figure 8, we present a summary in terms of the depicted changes caused by various factors, such as the oxidation state of copper (Cu(I) vs Cu(II)), the type of interface (solid/ aqueous vs solid/air), the incubation time, and the structure of  (1) 1-PBA interacts with the surface of CuONSs via only one aromatic ringthe phenyl ring, aligned at an angle to this surface, and lone pairs of electrons on the nitrogen and oxygen atoms of the −C(N)PO− fragment. At the Cu 2 ONP/water interface, the phosphonic acid group is moved away from the substrate surface and two aromatic rings of 1-PBA participate in the interaction with this substrate; the Ph ring adopts a vertical orientation with respect to the substrate surface, while the Ph B(OH) 2 ring is almost horizontal. The change of the interface from Cu 2 ONPs/water to CuO 2 NPs/air forces the molecule to "straighten up" so that the contact between the Ph B(OH) 2 ring and the substrate surface is maintained while the Ph ring moves away from this surface.
(2) By replacing the phenyl group with the benzyl group, the 2-PBA molecule interacts with the CuONS surface via the phosphonic acid group and the Ph ring, which moves away from the substrate surface and assumes a nearly vertical orientation with respect to that surface. 2-PBA adsorbs at the Cu 2 ONP/water and CuO 2 NP/air interfaces via the vertical Ph ring and the fragment −C(N)PO−, respectively.
(3) Further modification of the 1-PBA structure leads to another change in the bis{1-PBA} adsorption mode. That is, bis{1-PBA} is planar aligned near the CuONS surface, while the more or less vertical Ph B(OH) 2 rings are preserved. On the other hand, immediately after adsorption at the Cu 2 ONP/water interface, Ph B(OH) 2 is in contact with this interface and adopts a vertical orientation. In the following minutes after adsorption (up to 10 min), a reorientation is observedthe molecule lies down on the interface so that its two Ph B(OH) 2 rings adopt a more or less horizontal orientation with respect to the interface. Unlike at the Cu 2 ONP/air interface, the skeleton of bis{1-PBA} adopts an angular orientation.