Towards the differential diagnosis of prostate cancer by the pre-treatment of human urine using ionic liquids


Potassium citrate tribasic monohydrate (K3C6H5O7.H2O, purity ≥ 99 wt%) was obtained from Sigma-Aldrich Chemical Co. (USA). Prostate Specific Antigen from human semen (purity ≥ 95%) was obtained from Sigma-Aldrich Chemical Co. Methanol (purity > 99.9%) was acquired from Fisher Scientific. Acetonitrile (purity > 99.7%) was supplied from Lab-Scan. The buffers required for the ILs synthesis, namely n-cyclohexyl-2-aminoethanesulfonic acid (CHES, purity > 99 wt%), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, purity > 99.5 wt%), 2-(N-morpholino)ethanesulfonic acid (MES, purity > 99 wt%), n-(tri(hydroxymethyl)methyl)glycine (Tricine, purity > 99 wt%) and 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES, purity > 99 wt%) were purchased from Sigma-Aldrich Chemical Co. The cation precursor, tetrabutylphosphonium hydroxide ([P4444][OH], 40 wt% in H2O) was supplied by Sigma–Aldrich Chemical Co. Tetramethylsilane (TMS, purity > 99.9 wt%) and deuterium oxide (D2O purity > 99.9 wt%) were obtained from Sigma–Aldrich Chemical Co. Purified water passed through a reverse osmosis and a Milli-Q plus 185 water purifying system was used in all experiments. For the quantification of PSA using the Blitz Pro System, Super Streptavidin biosensors acquired from VWR were used.

Biological samples

Urine samples were provided by a healthy male donor after signing an informed consent according to the Declaration of Helsinki. The collection of urine samples was approved by the Ethics Committee of Cova da Beira University Hospital Center (CE 41/2015). The collection of data complied with the General Data Protection Regulation (Reg. (EU) 2016/679 and all procedures were carried out in accordance with relevant guidelines and regulations.

Synthesis and characterization of the Good buffers-ionic liquids (GB-ILs)

The GB-ILs ([P4444][MES], [P4444][TES], [P4444][CHES], [P4444][HEPES] and [P4444][Tricine]—chemical structures and complete descriptions given in in Supporting Information) were synthesized via neutralization of the base with the appropriate acid, according to previously reported protocols26. Briefly, a slightly excess of an equimolar aqueous solution (1:1.1) of buffer was added drop-wise to the tetrabutylphosphonium hydroxide solution. The mixture was stirred continuously for at least 12 h at room temperature (≈ 25 °C) to produce the IL and water as by-product. The mixture was then subjected to 50–60 °C under reduced pressure, resulting in a viscous liquid. A mixture of acetonitrile and methanol (1:1, v:v) was added and vigorously stirred at room temperature for 1 h. The solution was then filtered to remove any excess buffer. The organic solvents were evaporated and the GB-IL products dried under vacuum for 3 days at room temperature. The water content in each GB-IL was measured by Karl-Fischer (KF) titration, using a KF coulometer (Metrohm Ltd., model 831)—data given in the Supporting Information. The chemical structures and purity of the GB-ILs were confirmed by 1H and 13C NMR spectroscopy (Bruker AMX 300) operating at 300.13 and 75.47 MHz, respectively, whose spectra are given in the Supporting Information. Chemical shifts are expressed in δ (ppm) using tetramethylsilane (TMS) as internal reference and D2O as deuterated solvent. The ILs synthetized in this work showed high purity levels without signs of decomposition.

Phase diagrams and tie-lines

To ascertain the mixture compositions required to form two-phase systems that can be used as extraction/concentration platforms, the binodal curve of each ABS were initially determined through the cloud point titration method17 at 25 °C (± 1 °C) and atmospheric pressure. Aqueous solutions of K3C6H5O7 at circa 60 wt % and aqueous solutions of the different ILs (≈ 80 wt%) were prepared and used for the determination of the binodal curves, followed by the drop-wise addition of water until the finding of the monophasic region. The opposite procedure also was carried out to better describe the binodal curves, particularly at the salt-rich region, which is of extreme relevance to deal with ABS as concentration strategies. The ternary system compositions were determined by weight quantification within ± 10–4 g. The experimental binodal curves at 25 °C were fitted by Eq. (1)27:

$$left[ {IL} right] = A{text{ exp}}left( {Bleft[ {salt} right]_{{}}^{0.5} } right) – left( {Cleft[ {salt} right]_{{}}^{3} } right)$$


where [IL] and [salt] are the IL and the salt weight fraction percentages, respectively, and A, B and C are constants obtained by the regression of the experimental data.

Tie-lines (TLs), i.e. the composition of each phase for a given initial mixture composition at the biphasic region, were determined through the solution of the following system of four equations (Eqs. (2) to (5)) with four unknown values ([IL]IL, [IL]salt, [salt]IL, and [salt]salt):

$$left[ {IL} right]_{salt} = Aexpleft[ {left( {Bleft[ {salt} right]_{IL}^{0.5} } right) – left( {Cleft[ {salt} right]_{IL}^{3} } right)} right]$$


$$left[ {IL} right]_{salt} = Aexpleft[ {left( {Bleft[ {salt} right]_{salt}^{0.5} } right) – left( {Cleft[ {salt} right]_{salt}^{3} } right)} right]$$


$$left[ {IL} right]_{IL} = frac{{left[ {IL} right]_{M} }}{alpha } – frac{1 – alpha }{alpha }left[ {IL} right]_{salt}$$


$$left[ {salt} right]_{IL} = frac{{left[ {salt} right]_{M} }}{alpha } – frac{1 – alpha }{alpha }left[ {salt} right]_{salt}$$


where the subscripts IL, salt and M represent the top and the bottom phases and the mixture composition, respectively. The parameter α is the ratio between the weight of the top phase and the weight of the overall mixture. The respective tie-line lengths (TLLs) were determined by Eq. (6):

$$TLL = sqrt {left( {left[ {salt} right]_{IL} – left[ {salt} right]_{salt} } right)^{2} + left( {left[ {IL} right]_{IL} – left[ {IL} right]_{salt} } right)^{2} }$$


Extraction and quantification of prostate specific antigen (PSA) using IL-based ABS

An initial screening on the several ILs performance and mixture compositions was carried out, particularly to appraise the ABS ability to extract PSA from aqueous solutions without protein losses. To this end, different ABS, involving all the prepared ILs, and two mixture compositions (40 wt% salt + 30 wt% IL + 30 wt% H2O and 40 wt% salt + 40 wt% IL + 30 wt% H2O) were studied to evaluate the effect of the concentration of the phase-forming components. Aqueous solutions of PSA at concentrations circa 50 ng mL−1 were used as the “water” added to each ABS. The used PSA concentration was chosen after optimization using the BLItz Pro system. This type of quantification was chosen since it allows also to address the PSA stability and activity, where only active PSA binds with PSA-antibody. After, a careful separation of the phases was performed and the amount of PSA in each phase was quantified using the BLItz Pro system equipment, by an external standard calibration method using protein concentrations ranging from 1 to 100 ng mL−1. The anti-PSA solution was prepared in sample diluent buffer (PBS) at 100 µg mL−1. Anti-PSA was immobilized onto streptavidin sensors, which were hydrated for 15 min before being used. The immobilized sensors were placed into the sample diluent buffer for 30 s to observe the dissociation of anti-PSA. The anti-PSA-immobilized sensor tips were placed into the sample diluent buffer (PBS) for 30 s to set the binding baseline. The sensors were then used to quantify biologically active PSA in the IL- and salt-rich phases of ABS. At least three independent ABS were prepared and 2 samples of each phase quantified.

The percentage extraction efficiency of PSA (({text{EE}}_{{{text{PSA}}}} {text{% }})) is the percentage ratio between the amount of PSA in the IL-rich aqueous phase to that in the total mixture, and is defined according to Eq. (7):

$$EE_{{{text{PSA}}}} % = frac{{w_{PSA}^{IL} }}{{w_{PSA}^{IL} + w_{PSA}^{Salt} }} times 100$$


where (w_{PSA}^{IL}) and (w_{PSA}^{Salt}) are the total weight of PSA in the IL-rich and in the salt-rich aqueous phases, respectively. In all systems the top phase corresponds to the IL-rich phase whereas the bottom phase is mainly constituted by the salt and water.

The percentage recovery efficiency of PSA (RYPSA%) is the percentage ratio between the amount of PSA in the IL-rich aqueous phase to that in the initial PSA aqueous solution, and is defined according to Eq. (8):

$$RY_{{{text{PSA}}}} % = frac{{w_{PSA}^{IL} }}{{w_{PSA}^{Total} }} times 100$$


where (w_{PSA}^{IL}) is the total weight of PSA in the IL-rich phase and (w_{PSA}^{Total}) is the total weight of PSA in aqueous solution.

For the samples in which a concentration step was applied, PSA was quantified by SE-HLC. After a careful separation of the ABS phases, both phases were analysed by SE-HPLC. Each phase was diluted at a 1:9 (v:v) ratio in a phosphate buffer solution before injection. A Chromaster HPLC (VWR Hitachi) was used. The SE-HPLC was performed with an analytical column Shodex Protein KW- 802.5 (8 mm × 300 mm). A 100 mM phosphate buffer + NaCl 0.3 M was run isocratically with a flow rate of 0.5 mL.min−1. The column oven and autosampler temperatures were kept at 25 °C and at 10 °C, respectively. The injection volume was 25 μL. The wavelength was set at 280 nm using a DAD detector. The obtained chromatograms were treated and analysed using the OriginPro8 software. Equations (7) and (8) were applied to determine the extraction efficiency and recovery efficiency of the studied ABS for PSA.

Lever-arm rule

The lever-arm rule was used to determine the weight percentages ratio of the coexisting phases in the respective phase diagrams for given mixture compositions. Several extractions were carried out at different compositions in the same TL, allowing to work with different concentration factors. A fixed and long TL was initially selected, and the lever-arm rule was used to determine the weight fraction of each phase-forming component (IL and K3C6H5O7) to be used in each extraction corresponding to a given concentration factor (CF). The CF is defined as the ability along the same TL to maintain the composition of each phase while varying only the volume and weight ratio of the phases. Several ternary mixtures were prepared within the biphasic region with the “theoretical” weight percentages of salt, IL and H2O/PSA, corresponding to CFs of 5, 20, 50, 100, 150, 200 and 250-fold. ABS were first prepared as a control without adding PSA, and once achieved the CF of 250-fold, the extractions were performed adding an aqueous solution of PSA at a concentration of 150 ng mL−1 (the cut-off value found in urine13). Each mixture was vigorously stirred, centrifuged for 10 min, and left to equilibrate at (25 ± 1)°C.

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