Polypyrrole Based Molecularly Imprinted Polymer Platform for Klebsiella pneumonia Detection

Pyrrole (98%; purchased from Sigma Aldrich), hydrochloric acid (HCl), and chloroform 99.5% (both purchased from Qualigens) were used for the synthesis of PPy. Propane 2-ol (99.7%; fisher scientific), acetonitrile (99.9%; Thomas baker), and anhydrous ferric chloride (FeCl₃) (98%; HI media) were used in this procedure. HCl, potassium ferrocyanide (K₄[Fe(CN)₆]), potassium ferricyanide (K₃[Fe(CN)₆]), sodium phosphate dibasic dehydrate (Na₂HPO₄), and sodium phosphate monobasic dehydrate (NaH₂PO₄) were obtained from Fisher Scientific. All the solutions were prepared in DI water obtained from the Millipore water purification system. Indium tin oxide (ITO) coated glass with 1.1 mm thickness and sheet resistance of 25 ohm cm⁻² purchased from Blazers (U.K) having a transmittance of 90%. For the electrochemical response study, the bacterial concentrations of different CFU values (1–10,0000 CFU) were prepared.

All cell strains were cultured on Mueller-Hinton (MH) agar plate at 37 °C in an incubator. Before the experiment, the primary and secondary cultures were prepared. For primary culture, a single colony of bacteria was added to 5 ml of broth media and left in an incubator at 37 °C for 24 h. For secondary culture, 50 μl of primary culture is added into 5 ml of media and left for 3 and a half hrs in the incubator at 37 °C. This fresh culture was rinsed three times using PBS by centrifuging at 5000 rpm and 4 °C. OD 600 was used to measure the cell density in a suspension.

The synthesis of MIP of K. pneumonia was done by the interfacial oxidative polymerization process by using ferric chloride as an oxidizing agent. For this process two solutions are prepared in different beakers, first one is 50 mM ferric chloride solution (200 ml) containing 1 M HCl (20 ml) in which 1 ml of 0.5 OD K. pneumonia bacterial cells are added to it (known as aqueous phase) and the second one is 1 M pyrrole added in 20 ml chloroform (known as organic phase). For the interfacial polymerization, these two solutions were mixed carefully and slowly by transferring the aqueous phase solution in the beaker having pyrrole monomer solution along the sidewalls of the beaker and kept undisturbed for 24 h at room temperature. After 24 h, the PPy polymer layer was formed in between the oil and aqueous phases. The oil and aqueous phases were pipetted out slowly and carefully to collect the PPy polymer containing K. pneumonia cells. The unreactive residue of pyrrole monomeric units and oxidant were removed by washing the precipitate with DI and ethanol simultaneously and the washed polymer was collected by filtration. The collected filtrate was divided into two parts: the first part was dried and stored as it is without any further washing and named as NIP + K. pneumonia and another part was sonicated for 12 h continuously followed by centrifugation at 5000 rpm for 20 min, washed with DI water and dried at 60 °C overnight which was named as MIP. For the synthesis of non-imprinted polymer (NIP) all the above steps were kept the same except the addition of K. pneumonia cells. The final products MIP and NIP were characterized by FTIR. For the electrode fabrication by electrophoretic deposition-1 mg of MIP, NIP, and NIP + K. pneumonia in 500 μl of isopropyl alcohol and 500 μl of acetonitrile was taken in different vials and sonicated for 4 h. The schematic diagram (Scheme 1) shows the overall synthesis and identification of bacteria based on MIP.

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For the fabrication of electrodes via EPD method ITO coated glass of 1.5 cm × 0.5 cm was used. For this acetonitrile was used as an electrolyte or conducting liquid. The ITO glass sheet was cleaned with ethyl alcohol and DI water, and hydrolyzed these sheets by taking H₂O₂, NH_3, and water solution in 1:1:5 volume ratios and placing the sheets in this solution at 70 °C for 1 h and dried at room temperature. For electrophoretic deposition of the electrode, dispersion of MIP, NIP was done in acetonitrile with Mg ion used as a catalyst. For an electrochemical reaction, a voltage of 50 V was applied. Due to the difference in potential between electrodes, ions created at the electrode migrated to the oppositely charged electrode, and the film was deposited on the ITO-coated glass sheet. The schematic diagram (Scheme 1) shows the overall synthesis and identification of bacteria based on MIP.

Figure 1 shows the FTIR spectrum of NIP and MIP samples. There are changes in the bond vibrations observed in MIP as compared to NIP as follows. In NIP the occurrence of a weak peak at 3400 cm⁻¹ is assigned to the presence of N–H stretching vibrations of the pyrrole ring. The weak band at 1800 cm⁻¹ is due to C–H stretching. The characteristic peak at 1600 cm⁻¹ corresponds to the C=C stretching and the peak at 1100 cm⁻¹ is due to C–C stretching confirming the formation of PPy. ³⁵ The FTIR spectra of MIP samples after removing bacterial cells are recorded the same as in NIP in the range of 4000 to 500 cm⁻¹ to confirm polymerization. All the peaks that are of NIP are matching in both cases. Although the peak positions are the same, there is a change in shape and intensity was observed. There are few extra peaks are also observed due to the interaction between PPy and lipopolysaccharide, peptide bonds present in the outer membrane of the bacterial cell. The peak we get around at 900 cm⁻¹, 1000 cm⁻¹ and 1100 cm⁻¹ which are due to C–H stretching and CH out of plane deformation, vinyl indene bond stretching, CN stretching in aromatic rings, and phosphorous oxy acids respectively, which were of few bacterial cells remained in MIP after removal.

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The pH value has a significant role in the electrochemical reaction between the analyte and electrode. Therefore, 0.2 mM PBS buffer solution having [Fe(CN)₆]^3−/4− redox species at different pH values varying from 6 to 8 pH was used to see the effect of pH by the DPV curve in the potential range from −0.8 to 0.8 on MIP/ITO electrode. As shown in Fig. 2a, the peak current changes with the change in pH value. More specifically, the electrochemical response of MIP gradually decreased as the pH increased from 6.5 to 7.4 and then increased to pH 8.0. This study showed that K. pneumonia cells (0.05 OD) interact more with imprinted cavities at pH 8.0 by showing higher DPV peak current values. Therefore, pH 8.0 buffer was used for further observations.

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The time required to bind the analyte molecule with the MIP surface depends on the incubation time. It has an important role in the performance of the sensor. Therefore, response time studies were analyzed by using DPV curve within the time interval 0 to 12 min, and the peak currents are plotted in Fig. 2b. This study was carried out with K. pneumonia bacteria cell (0.05 OD) added to the buffer in an electrochemical cell containing MIP and the DPV peak current was noted after every 3 min. It was observed that the peak current decreased at 3 min and later there is no significant change in the current. Therefore, it was concluded that approx. 3 min is required for the interaction of the bacterial cell with the MIP electrode. Thus, every reading of the response study after introducing the bacterial cell was taken after 3 min.

To analyze the electroactivity of NIP + bacteria/ITO, MIP/ITO, and NIP/ITO electrodes, the CV (at scan rate 50 mV s⁻¹) and DPV studies in the potential range of −0.4 V to 0.6 V were carried out. Figures 3a and 3b showed the CV and DPV current response of (i) NIP + bacteria/ITO, (ii) NIP/ITO and (iii) MIP/ITO electrodes. It was observed that the peak current of NIP/ITO and NIP with bacteria/ITO have almost the same current with a slight increase in the peak voltage. Whereas, in MIP (iii), the peak current was increased It was observed that the current increased drastically for MIP/ITO electrode after the bacterial cell was removed from the polymer matrix by simply washing and ultrasonication. This may be due to the formation of cavities on PPy after the removal of bacterial cells. So, the electroactivity of the electrode increased and thereby increasing the CV and DPV peak currents of MIP. These changes in the peak current and FTIR study showed that bacterial cell was successfully removed from the polymer matrix in MIP.

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The electrokinetics behavior of MIP/ITO electrodes was analyzed by the CV study by changing the scan rate from 10 to 100 mV s⁻¹. This study gives an idea about the kinetics of the reaction which occurs at the interface of the electrode and electrolyte solution (Fig. 3c). Between the peak current Ipa (anodic) and Ipc (cathodic) to the square root of √v there is a linear relationship observed as shown in the inset of Fig. 3c which indicates the diffusion-controlled electrochemical process which depends on the diffusion of electrolyte species. ^36,37

The Ipa/Ipc ratio of the MIP electrode was found to be 0.80 indicating the irreversible electron transfer kinetics for MIP/ITO electrode ^36,38 in the medium. The interface kinetic parameters of the electrodes such as surface area (Ae), the electron transfer coefficient (Ks) [calculated using Eq. 1], the surface concentration of absorbed electroactive species (γ*) [calculated using Eq. 2], Diffusion coefficient (D) [calculated using Eq. 3], were estimated to analyze the movement of electrons. The Randles-Sevcik equation (Eq. 3) determined the diffusion coefficient (D) as mentioned in the study of Lakshmi et al. ³⁹

$$\begin{eqnarray}&&{\rm{Ks}}=\mathrm{mnFV}/\mathrm{RT}\end{eqnarray} \tag{ 1 }$$

$$\begin{eqnarray}&&{\gamma }^{\ast }=4{{\rm{RTI}}}_{{\rm{a}}}{/n}^{{\rm{2}}}{{\rm{F}}}^{{\rm{2}}}{\rm{AV}}\end{eqnarray} \tag{ 2 }$$

$$\begin{eqnarray}&&{\rm{D}}={{\rm{I}}}_{a}^{2}/{\left(2.99\times {10}^{5}\right)}^{2}{A}^{2}{n}^{3}{C}^{2}V\end{eqnarray} \tag{ 3 }$$

Where n = 1 for ferri-ferro buffer, F = 96485, R = 8.314,T = 300 K,V = 50 mV s⁻¹.

C = concentration of buffer = 0.1 M.

A = area of the electrode.

m = E_a-E_c.

The obtained values are Ks = 0.519, γ* = 1.734*10⁻⁸, D = 1.46*10⁻¹². The results show that there exists good conductivity for charge transfer between the electrolyte and the electrode.