Fluorescent Nile blue‐functionalized poly (N‐isopropylacrylamide) microgels responsive to temperature and polyamines

Fluorescent poly(N‐isopropylacrylamide‐co‐Nile blue) (pNIPAm‐co‐NB) microgels were synthesized that exhibited fluorescence intensity changes in a water temperature‐dependent fashion. NB is well known to exhibit fluorescence intensity that depends on the hydrophobicity of the environment, while pNIPAm‐based microgels are well known to transition from swollen (hydrophilic) to collapsed (relatively hydrophobic) at temperatures greater than 32 °C; hence, we attribute the above behavior to the hydrophobicity changes of the microgels with increasing temperature. This phenomenon is ultimately due to NB dimers (relatively quenched fluorescence) being broken in the hydrophobic environment of the microgels leading to relatively enhanced fluorescence. We went on to show that the introduction of cucurbit[7]uril (CB[7]) into the pNIPAm‐co‐NB microgels enhanced their fluorescence allowing them to be used for polyamine (e.g., spermine [SPM]) detection. Specifically, CB[7] forms a host–guest interaction with NB in the microgels, which prevents NB dimerization and enhances their fluorescence. When SPM is present, it forms a host–guest complex that is favored over the CB[7]‐NB host–guest interaction, which frees the NB for dimerization and leads to fluorescence quenching. As a result, we could generate an SPM sensor capable of SPM detection down to ~0.5 µmol/L in complicated matrixes such as serum and urine.

polymeric hydrogels useful for biological applications, for example, bioimaging and disease diagnosis. 9,10Stimuliresponsive fluorescent microgels are hydrogel particles containing fluorescent moieties, such as quantum dots, fluorophore dyes, and fluorescent conjugated polymers. 9he stimuli-responsivity of the microgels allows them to change their fluorescence properties when exposed to external stimuli.In addition, the polymeric network can improve the photostability of fluorophores by providing efficient screening of fluorophores from oxygen and radical attack. 11oly(N-isopropylacrylamide) (pNIPAm) is a thermoresponsive polymer that has been investigated extensively over the past few decades. 1 Linear pNIPAm exhibits a lower critical solution temperature (LCST) at ~32 °C, where it undergoes a reversible coil (extended) to globule (collapsed) transition. 12That is, when the solution temperature is below the LCST, the pNIPAm chain exists as a "solvated" random coil, and transitions to a "desolvated" globule as the temperature exceeds the LCST.The same thermoresponsive properties are found in pNIPAm-based microgels.PNIPAm-based microgels are highly swollen in water because the pNIPAm network chains are hydrophilic at T < LCST; whereas they collapse (expel water) and become relatively hydrophobic at T > LCST. 13olyamines (e.g., spermine [SPM]) are essential for cell growth, proliferation, immune response, neuron regulation, and the synthesis of proteins and nucleosides. 14,15In addition, polyamines protect DNA, proteins, and lipids from the damage of reactive oxygen species. 16,17Recent studies showed that SPM could be used as a biomarker for many diseases, such as psoriasis, peptic ulcers, and chronic gastritis. 18Due to the involvement of SPM in cell growth and proliferation, SPM can also serve as a cancer biomarker for monitoring the growth of tumors in various types of cancer. 19herefore, the detection of the level of SPM in biofluids can be very important.Traditional methods, such as immunoassay and chromatography combined with mass spectrometry, have previously been applied for the detection of SPM. 20,21However, these methods are time-consuming and require expensive equipment as well as skilled personnel.
Over the past few years, several hydrogel-based strategies have emerged for the detection of SPM.For example, Ratish et al. 15 developed a novel dye 3-((7-hydroxy-4-methylcoumarin)methylene)aminophenylboronic acidincorporated agarose hydrogel for the detection of SPM and spermidine (SPD).This hybrid hydrogel exhibited a fluorescence response in the presence of SPM and SPD with a limit of detection (LOD) of 6 μmol/L.In another example, Ikeda et al. 22 used a montmorillonite supramolecular hydrogel with a cationic fluorophore embedded that could be used for SPM sensing in the 20-100 μmol/L concentration range.However, the LOD of the sensor in urine is higher than the SPM concentration in patients with blood and solid tumors (8-10 μmol/L). 23In addition to fluorescent dye-incorporated hydrogel-based sensors, Quan et al. 24 fabricated an Eu(III) ion-functionalized crystalline polyimide hydrogel film, which exhibited enhanced fluorescence intensity as the concentration of SPM in serum increased, showing a LOD of 8.2 μmol/L.Based on the reported analytical figures of merit, it is clear that there is still room for the development of novel techniques for detecting biologically relevant concentrations of SPM.
In this study, we synthesized fluorescent pNIPAm-co-Nile blue acrylamide (pNIPAm-co-NB) microgels by utilizing the monomer Nile blue acrylamide (NBA), and the crosslinker N, N′-methylenebis(acrylamide) (BIS).NB is a typical enzophenoxazine-based fluorescent dye, which can partially form nonfluorescent NB dimers and thus, emits weak blue fluorescence in an aqueous solution. 25,26As mentioned above, temperature changes can trigger the swelling and deswelling of pNIPAm microgels, which affects the fluorescence of the microgel.Specifically, as the temperature increases, pNIPAmbased microgels collapse above the LCST, leading to a more hydrophobic environment.The hydrophobic environment is known to increase NBs fluorescence quantum yield since the electrostatic repulsion between NB is stronger in the hydrophobic environment compared to the hydrophilic environment, 27 causing the nonfluorescent NB dimers to break and thus enhancing the fluorescence.Furthermore, cucurbit [7]uril (CB [7]) was added to the pNIPAm-co-NB by exploiting the CB [7]-NB host-guest interaction, which prevented NB dimer formation and exhibited a strong blue fluorescence.We finally used pNIPAm-co-NB/CB [7] microgels to sense SPM using fluorescence, since SPM can compete with the NB moieties to bind to CB [7] molecules and remove the bound CB [7] from pNIPAm-co-NB microgels, resulting in the regeneration of NB dimers and thus decreasing the fluorescence of the microgel.It was found that our sensors can detect the SPM concentration down to ~0.5 μmol/L in serum and urine matrixes with a linear range of 0.2-1000 μmol/L.

| Synthesis of pNIPAm-co-NB microgels and pNIPAm microgels
pNIPAm-co-NB microgels were synthesized via free radical precipitation polymerization.NIPAm (18.98 mmol), NBA (0.02 mmol), and BIS (1 mmol) were dissolved in DI water (99 mL) in a beaker and stirred for 20 min.A 0.2 μm filter affixed to a 20 mL syringe was used to filter the solution, and the filtered solution was transferred into a 250 mL three-neck round-bottom flask.The flask was fitted with a thermometer, a condenser, and an N 2 gas inlet (via a needle).The solution was stirred at 500 r/min and bubbled with N 2 gas while increasing the temperature to 70 °C over 1 h.Afterward, the surfactant CTAB (0.01 mmol) was added to the solution, followed by the introduction of the initiator V50 (0.2 mmol) dissolved in 1 mL of DI water.The polymerization reaction was carried out at 70 °C for 4 h under an N 2 atmosphere.The suspension was allowed to cool down to room temperature with stirring and filtered through a Whatman #1 filter paper to remove large aggregates.The suspension was centrifuged at 10,000 r/ min for 1 h to form a pallet, and the supernatant was discarded and replaced with DI water for resuspension.The process was repeated eight times to remove unreacted monomers and linear polymers until the supernatant became colorless.
pNIPAm microgels, without NBA, were synthesized, using the same process as described above.After the polymerization reaction, 0.02 mmol of NBA was added to the resulting microgel solution, stirring for 4 h.Then, the microgel solution was filtrated and purified via centrifugation, as mentioned above.

| Temperature responsivity of pNIPAm-co-NB microgels
The lyophilized pNIPAm-co-NB microgel powder was dissolved in DI water to prepare a 0.5 mg/mL pNIPAmco-NB microgel solution.The microgel solution was placed in a 96-well plate, and its fluorescence was measured at various temperatures, using a SpectraMax i3x microplate reader (Molecular Devices).The fluorescence intensity at 674 nm was measured with an excitation wavelength of 635 nm.
A UV-Vis spectrometer (Hewlett-Packard Agilent 8453) coupled with an 89090A temperature controller was used to measure the absorbance at 480 nm of pNIPAm-co-NB microgel solution (50 μg/mL) at various temperatures.

| Synthesis of CB[7]
CB [7] was prepared by a modified procedure from the previous paper. 28,29Specifically, glycoluril (15 g, 0.105 mol) was dissolved in 75 mL of ice-cold concentrated HCl in a 250 mL round bottom flask.Then, paraformaldehyde (6.3 g, 0.21 mol) was added to the solution, which formed a gel immediately.The gel mixture was heated to 90 °C under vigorous stirring, and the gel turned into solution again at ~60 °C.After 1 h of heating, the solution was heated at 100 °C for 18 h.The reaction mixture was poured into 400 mL of acetone, and the resultant precipitate was collected by vacuum filtration.Next, the precipitate was dissolved in 300 mL of 20% aqueous glycerol and heated at 80 °C for 4 h, and the insoluble solid was filtered out via vacuum filtration.The resulting filtrate was treated with 300 mL of methanol to generate CB [7] precipitate that was collected via vacuum filtration.Finally, the collected CB [7] solid was washed with methanol three times and dried under vacuum.The 1 H NMR spectrum of the synthesized CB [7]  (Supporting Information S1: Figure S1) shows the characteristic peaks of CB [7], 30 which suggests the CB [7] was synthesized successfully.

| Characterization
Transmission electron microscopy (TEM) images were taken using a JEOL JEM-ARM200CF S/TEM electron microscope (JEOL USA, Inc) at an accelerating voltage of 200 kV.NMR spectra were recorded, using an Agilent VNMRS four-channel, dual receiver 700 MHz spectrometer.The hydrodynamic size and Zeta potential of the microgel were measured by dynamic light scattering (DLS), using a Zetasizer Nano ZS-Malvern Instrument equipped with a light source with a wavelength of 633 nm.For the hydrodynamic size measurement, the pNIPAm-co-NB microgel sample was prepared in a phosphate buffer (1 mmol/L, pH 6.0) with a concentration of 0.1 mg/mL.The pNIPAm-co-NB with CB [7]  solution was prepared by spiking 100 μmol/L CB [7] to the above 0.1 mg/mL microgel solution.The pNIPAm-co-NB with CB [7] and SPM solution was prepared by spiking 100 μmol/L SPM to the previous CB [7]-microgel solution.Finally, the pNIPAm-co-NB with SPM solution was prepared in a phosphate buffer (1 mmol/L, pH 6.0) with 100 μmol/L SPM and 0.1 mg/mL microgel.For the zeta potential measurement, 0.5 mg/mL pNIPAm-co-NB and pNIPAm microgels were dissolved in DI water and the pH was adjusted to 6.0, using HCl.

| Characterization of pNIPAm-co-NB microgels
pNIPAm-co-NB microgels were prepared using free radical precipitation polymerization.Due to the incorporation of the NB moiety, the pNIPAm-co-NB microgel solution exhibited a green color (Figure 1A).As a simple control, if we mixed pNIPAm microgels with the NBA monomer, followed by the same purification procedure used for the pNIPAm-co-NB microgels, the pNIPAm microgel solution was white (Figure 1A), indicating that NBA cannot effectively be captured in the microgels via noncovalent interactions.Furthermore, 1 H NMR was used to characterize the pNIPAm-co-NB microgels.As can be seen in the spectrum in Figure 1A, peaks at 7-8 and 1.19 ppm can be assigned to the aromatic protons of NBA and methyl protons of NIPAm, respectively. 31The absence of vinyl proton peaks at 6-7 ppm demonstrated that monomers do not exist in the microgel (Supporting Information S1: Figure S2).Therefore, it can be concluded that pNIPAm-co-NB microgels were successfully prepared.The TEM images in Figure 1B showed that the pNIPAmco-NB microgels had a spherical morphology, with a dry diameter of ~550 nm, which is very similar to the morphology and diameter of pNIPAm microgels (Supporting Information S1: Figure S3A).Meanwhile, the average hydrodynamic diameter of the microgels in DI water was ~750 nm (PDI = 0.135).Compared to pNIPAm microgels, pNIPAm-co-NB microgels showed an increase in Zeta potential from ~0 to ~6 mV (Figure 1C), which was attributed to the positive charges on the NB moieties of the microgel.In addition, we measured FTIR spectra of pNIPAm, pNIPAm-co-NB microgel, and NBA monomer (Supporting Information S1: Figure S4).Since NIPAm is the primary monomer of pNIPAm and pNIPAm-co-NB microgels, their FTIR spectra show the same characteristic bands around 3300, 1650, and 1540 cm −1 , which are attributed to the symmetric stretching of the N-H bond, the stretching of the amide C═O, as well as the bending of N-H, respectively. 32,33The NBA monomer absorption bands are not observed in the FTIR spectrum of pNIPAmco-NB microgel due to the low abundance of NBA monomer (~0.1%) in the microgel.It is worth noting that the FTIR spectrum of pNIPAm-co-NB exhibits two distinct absorption bands at 1172 and 1130 cm −1 compared to the FTIR spectrum of pNIPAm, which seems to match the absorption bands at 1166 and 1121 cm −1 in the NBA sample.Since the other absorption bands of NBA are not observed, those bands may not come from the NB moiety of the microgel.

| Temperature responsitivity of pNIPAm-co-NB microgels
As a typical enzophenoxazine-based fluorescent dye, NB has a stable and rigid conformation and conjugated system, emitting blue fluorescence. 25As shown in Supporting Information S1: Figure S5, the control pNIPAm microgel solution does not fluoresce under UV light, while pNIPAm-co-NB exhibits a greenish fluorescence under UV light.We used fluorescence spectroscopy to explore the optical properties of pNIPAm-co-NB microgels.It was found that the maximum excitation/emission wavelength of the pNIPAm-co-NB microgel was 635/674 nm (Figure 2).Compared to the NBA dye itself, the F/F 0 of pNIPAm-co-NB decreased more slowly over time (Supporting Information S1: Figure S6), indicating that the polymeric network can significantly enhance the photostability of the NB dye.
Furthermore, we studied the thermoresponsivity of pNIPAm-co-NB microgels by monitoring the change in fluorescence intensity of the microgel at 674 nm (I 674 , λ ex = 635 nm) as a function of increasing temperature.As shown in Figure 3, I 674 increased with increasing temperature from 25 °C to 45 °C, showing the apparent LCST at 37 °C.This trend can be explained due to the microgels expelling their solvating water (and collapsing) at elevated temperature, resulting in NB dimer disassembly, and enhanced fluorescence, as shown in Scheme 1.By the microgels shrinking, the NB molecules come closer together resulting in increased electrostatic repulsion, and a more hydrophobic environment, leading to the hindrance of NB dimer formation. 25s a comparison, the thermoresponsive behavior of the pNIPAm-co-NB microgels was also investigated utilizing a UV-Vis spectrometer by monitoring light scattering of the microgels in solution as a function of increasing temperature.It is important to note that for these experiments absorbance is proportional to scattering-the UV-Vis is simply being used to probe microgel solvation state.As can be seen in Figure 3, the absorbance (i.e., scattering) of the pNIPAm-co-NB microgels at 480 nm exhibited an increase in scattered light intensity with an increase in solution temperature from 20 °C to 50 °C, due to the microgels collapsing (and becoming more optically dense) at elevated temperature.We note that the scattered light intensity tracks the fluorescence intensity measurements nearly perfectly, meaning that the desolvated (and hydrophobic) microgels lead to enhanced fluorescence.However, we did note that the LCST value measured from fluorescence (37 °C) was higher than that from UV-Vis tests (33 °C), which was possibly due to the initial shrinkage of the microgel not being hydrophobic enough to cause the NB dimers to switch to the more fluorescent monomeric form.

| SPM responsitivity of pNIPAm-co-NB microgels
For the purpose of SPM sensing, we first mixed pNIPAmco-NB microgels with CB [7], forming pNIPAm-co-NB/CB [7] microgels.The morphology of pNIPAm-co-NB/CB [7]  did not show any difference from the pNIPAm-co-NB microgels, as shown in Supporting Information S1: Figure S3B.Compared to the FTIR spectra of pNIPAmco-NB microgel, the FTIR spectra of pNIPAm-co-NB/CB [7] microgel showed an additional band at 1725 cm −1 , which was attributed to the stretching of C═O on CB [7]  molecules (Supporting Information S1: Figure S7).After adding CB [7], the fluorescence of pNIPAm-co-NB/CB [7]   F I G U R E 2 Excitation (blue dashed line) and emission (red solid line) spectra of the fluorescent poly(N-isopropylacrylamideco-Nile blue) microgel solution.microgel solution exhibited a strong blue emission (Supporting Information S1: Figure S5).As seen in Figure 4, the relative fluorescence intensity (F/F 0 ) of the pNIPAm-co-NB/CB [7] microgels increased as the concentration of CB [7] increased from 0 to 500 μmol/L, where F/F 0 refers to the I 674 at a given concentration of CB [7] divided by that at 0 μmol/L CB [7].Macrocyclic CB [7] possesses a cavity that exhibits a strong affinity for the protonated amines of NB via ion-dipole interactions, especially when the substrate is a good fit for the cavity. 34s a consequence, the presence of CB [7] prevents the formation of nonfluorescent NB dimers and the interactions between NB and water molecules, resulting in the enhancement of F/F 0 .In the presence of SPM, the host-guest complex between CB [7] and SPM is formed, because the binding constant (K A ) between SPM and CB [7] (2.0 × 10 6 L/mol) is an order of magnitude larger than that between NB and CB [7] (2.5 × 10 5 L/mol, Supporting Information S1: Figure S8).Therefore, CB [7] is removed from the NB, freeing up the NB to form dimers, causing the fluorescence of the microgel solution to decrease (Scheme 2).
The DLS results (Supporting Information S1: Figure S9) show that the hydrodynamic diameter of the microgel slightly increased from 764 to 829 nm after adding CB [7], indicating that the fluorescence enhancement was not due to the shrinkage of the microgel.The size increase can be rationalized by accounting for the hydrophilic exterior of CB [7] causing the microgel to become more hydrated.Moreover, the microgel is expected to become less crosslinked (and more hydrophilic) as the NB dimer was broken by CB [7] binding, resulting in the increase in microgel hydrodynamic diameter (and hydroohilicity), and what should be a relatively quenched fluorescence.Clearly, the breaking of the NB dimers is the dominant factor dictating the fluorescence intensity.After adding SPM, the hydrodynamic diameter of the CB [7]-microgels decreased from removed from the microgel's NB groups by forming CB [7]-SPM complexes, allowing the NB dimers to be reformed, and the fluorescence quenched.In addition, the SPM has little impact on the hydrodynamic size of the microgel itself (Supporting Information S1: Figure S9).Moreover, the microgel fluorescence was not affected by the addition of SPM without the presence of CB [7], as shown in Supporting Information S1: Figure S10.
To optimize the performance of the SPM sensor, 1 mg/mL of the pNIPAm-co-NB microgels was first mixed with three concentrations of CB [7] (50, 100, and 200 μmol/L) in Na 2 HPO 4 /HCl buffer (1 mmol/L, pH 6.0) at ~22 °C.As can be seen in Figure 5, we observed that F/ F 0 generally decreased as the SPM concentration increased due to the regeneration of nonfluorescent NB dimers caused by the removal of CB [7] from the microgels (Figure 5).More specifically, in the case of 50 and 100 μmol/L of CB [7], pNIPAm-co-NB/CB [7]  exhibited a decrease in F/F 0 when the SPM concentration was above 1 μmol/L, while the initial decrease in F/F 0 occurred at 5 μmol/L of SPM in the case of 200 μmol/L of F I G U R E 4 Relative fluorescence (F/F 0 ) of the poly(Nisopropylacrylamide-co-Nile blue) (pNIPAm-co-NB) microgel solution as a function of increasing cucurbit [7]uril concentration.The data were obtained by averaging three measurements, and the error bars represented the standard deviation.
S C H E M E 2 Illustration of the sensing mechanism for the detection of spermine using poly(N-isopropylacrylamide-co-Nile blue) microgel and cucurbit [7]uril.
CB [7].The delayed response could be attributed to the initial binding of SPM with excess free CB [7] molecules.Although the microgel solution containing 50 μmol/L of CB [7] was very sensitive to the low concentration of SPM (0.5-10 μmol/L), it had a narrow sensing range of up to 10 μmol/L of SPM.On the contrary, the microgel solutions containing 100 μmol/L of CB [7] can sense SPM from 0.5 to 500 μmol/L.Hence, 100 μmol/L of CB [7]  was chosen for the detection of SPM.In addition, we compared the sensor performance for SPM detection at a "low" temperature (25 °C) and a "high" temperature (45 °C), as shown in Supporting Information S1: Figure S11.Compared to the low-temperature results, the addition of SPM at a high temperature (45 °C) did not lead to a decrease in the fluorescence intensity of the pNIPAm-co-NB/CB [7] solution.When the temperature is above the LCST of the microgel, the microgel environment becomes more hydrophobic and most NB moieties are in their monomer form, resulting in enhanced fluorescence.Because of this, the addition of CB [7] and SPM has less contribution to the breaking and reformation process of NB dimers.Consequently, CB [7] and SPM have little impact on the fluorescence intensity.
We further examined the performance of the pNIPAm-co-NB microgel-based SPM sensor in diluted human serum and urine.For comparison, the performance of the sensor in phosphate buffer was taken from the previous CB [7] optimization experiment with 100 μmol/L CB [7].For the serum and urine sample, we spiked various concentrations of SPM into five-fold diluted serum and urine.Then, we mixed the CB [7]  microgel solution with the SPM-spiked serum and urine solutions at a 1:1 volume ratio; thus, the resulting solutions containing 10-fold diluted serum and urine.As shown in Figure 6A, the F/F 0 decreased as the SPM concentration increased for buffer, serum, and urine samples.Overall, the responses of the pNIPAm-co-NB microgel sensors to SPM were very similar in all three media.Especially at the low SPM concentration range (0.5-5 μmol/L), the relative fluorescence intensities of the three media were almost exactly overlapped.For the buffer, the fluorescence intensity was fitted linearly over the SPM concentration of 0.5-500 μmol/L, with an equation of y = −0.115log(x) + 1.012 (R 2 = 0.98822) and a lower LOD of 0.6 μmol/L (Figure 6B).For the serum, the fluorescence intensity showed a linear relationship with SPM concentration between 0.5 and 1000 μmol/L, with an equation of y = −0.094log(x) + 1.034 (R 2 = 0.98814) and a lower LOD of 0.6 μmol/L (Figure 6C).A linear relationship was found in the urine spiked with SPM as well.The linear range was over the SPM concentration of 0.5-1000 μmol/L, with an equation of y = −0.096log(x) + 1.01 (R 2 = 0.97703), and the lower LOD was 0.5 μmol/L (Figure 6D).
It is known that the SPM concentration in blood is increased in psoriasis patients (~7.23 μmol/L) compared to that of healthy people (~2.53 μmol/L). 35Also, elevated SPM concentration in urine (8-10 μmol/L) can be observed in patients with blood and solid cancers, while the normal concentration of SPM in urine is ~1 μmol/L. 23he above result indicated that the pNIPAm-co-NB microgel sensor could be used to detect abnormal levels of SPM in either serum or urine samples.Compared to traditional chromatography and immunoassay-based techniques, our sensor is rapid and cost-effective.36][37][38][39] To explore the selectivity of our pNIPAm-co-NB microgel-based sensors for SPM detection, we performed an interference study in different cations and anions.As shown in Figure 7, the relative fluorescence change (F 0 − F/F 0 ), where F 0 and F refer to the I 674 of pNIPAmco-NB/CB before and after adding SPM or different ions, respectively, was much stronger in SPM than other ions, indicating that cations and anions were not able to interference with the SPM detection.Although cations (e.g., K + , Na + , and Ca 2+ ) are capable of binding to CB [7]  molecules through the ion-dipole interaction, the binding affinity of those cations is too low compared to that of F I G U R E 5 Relative fluorescence (F/F 0 ) of the cucurbit [7]uril (CB [7]) and poly(N-isopropylacrylamide-co-Nile blue) microgel solution as a function of spermine concentration.Three concentrations of CB [7] were added to the microgel solution: 50 μmol/L (red), 100 μmol/L (green), and 200 μmol/L (blue).The data were obtained by averaging three measurements, and the error bars represented the standard deviation.SPM. 34Therefore, the cations and anions had very little impact on the detection of SPM.
Furthermore, we tested the selectivity of our sensor toward biogenic amines, including SPD, putrescine (Put), Lys, Arg, His, Gly, and DA due to the potential interference from the binding between their positively charged amine groups and CB [7]. Figure 8 shows that the sensor had no significant response to the other biogenic amines, except for SPD.
SPD (SPM precursor) can be converted to SPM via aminopropyl group addition after its reaction with SPM synthase.The three positively charged amines make SPD a great encapsulation guest for CB [7].Supporting Information S1: Figure S12 showed that the binding constant between SPD and CB [7] was determined to be 1.1 × 10 6 L/mol, which was larger than the binding constant between NBA and CB [7].Therefore, SPD can bind to CB [7] through a competitive replacement of the NB moieties of pNIPAm-co-NB microgel, leading to a fluorescence decrease in the microgel solution.The normal levels of SPD in urine are ~1.2 μmol/L, similar to the normal urinary level of SPM, while the urinary concentration of SPD and SPM in cancer patients increases to ~26 and ~8 μmol/L, respectively. 23,46Since the elevation of SPM and SPD concentrations often occurs simultaneously, the abnormal polyamine (SPM and SPD) concentration still can be detected, using our pNIPAm-co-NB microgel-based sensor.

| CONCLUSION
In this study, fluorescent pNIPAm-co-NB microgels were synthesized via precipitation polymerization, using NI-PAm and NBA monomers as well as the crosslinker BIS.With the thermoresponsive pNIPAm backbone, the microgel exhibited thermoresponsive behavior, and its fluorescence decreased with increasing temperature.The increase in the hydrophobicity of the microgel environment in response to the increasing temperature may cause the disruption of the dimer form of the NB, resulting in enhanced fluorescence.By introducing CB [7]   molecules, the pNIPAm-co-NB microgel was capable of measuring the SPM concentration in serum and urine.The binding between CB [7] and the NB moieties of the   microgel could increase the fluorescence because the CB [7] encapsulation could prevent NB dimerization.The addition of SPM could disassemble the NB-CB [7]  complex via a competitive replacement of NB from CB [7] cavity by SPM, which resulted in the regeneration of NB dimers and a decrease in fluorescence.Finally, the pNIPAm-co-NB microgel-based sensor showed a great selectivity toward various cations and anions, and it did not show a response to many biogenic amines except for SPD which is a precursor of SPM.
1 H NMR spectrum of the poly(N-isopropylacrylamide-co-Nile blue) (pNIPAm-co-NB) microgel in D 2 O. Inset shows digital images of (i) pNIPAm-co-NB and (ii) pNIPAm microgel solutions.Concentration: 10 mg/mL.(B) Hydrodynamic diameter of the pNIPAm-co-NB microgel.The inset is the transmission electron microscopy image of the pNIPAm-co-NB microgel.The hydrodynamic size results were obtained by averaging three measurements, and the error bar represented the standard deviation.(C) Zeta potential of the pNIPAm-co-NB and pNIPAm microgels.
S C H E M E 1 Illustration of the thermoresponsivity, and concomitant fluorescence quenching and enhancement of poly(Nisopropylacrylamide-co-Nile blue) microgels.F I G U R E 3 Change in UV-Vis absorbance (left axis) at 480 nm and fluorescence intensity (right axis) of poly(N-isopropylacrylamide-co-Nile blue) microgel solution as a function of increasing temperature.The data were obtained by averaging three measurements, and the error bars represented the standard deviation of the measurements.

F I G U R E 6
The performance of the poly(N-isopropylacrylamide-co-Nile blue) (pNIPAm-co-NB) microgel-based spermine (SPM) sensor in phosphate buffer, serum, and urine.Relative fluorescence (F/F 0 ) of the pNIPAm-co-NB microgel solution as a function of SPM concentration in phosphate buffer, serum, and urine (A).The linear range was fitted for buffer (B), serum (C), and urine (D).The data were obtained by averaging three measurements, and the error bars represented the standard deviation.
Comparison of different analytical methods for spermine detection.
T A B L E 1