Lanthanide chelate-encapsulated polystyrene nanoparticles for rapid and quantitative immunochromatographic assay of procalcitonin

Abstract

Procalcitonin (PCT) is a potentially specific early marker of bloodstream infection and sepsis. In this work, we report, for the first time, the development of a fluorescence-based immunochromatographic strip test (IST) which employs Eu(DBM)₃phen-containing nanoparticles as reporters for the detection of PCT in human serum. Experimental results demonstrate that the developed IST has an excellent ability for rapid (15 min), sensitive (0.05 ng mL⁻¹) and quantitative analysis of PCT, and the assay results are comparable to which of the conventional enzyme-linked immunosorbent assay (ELISA). These results suggest that the developed assay could be utilized for point-of-care detection of PCT.

---

Introduction

Procalcitonin (PCT) is a small protein composed of 116 amino acids with a molecular weight of 13 kDa. It is a well-established, potentially specific early marker of bloodstream infection and sepsis, particularly in children.¹ It was also widely recognized as a prognostic and therapeutic indicator of bacteremia.² The commonly employed diagnostic method for analysis of PCT includes enzyme-linked immunosorbent assay (ELISA),³ chemiluminescence immunoassay (CLIA) and electro-chemiluminescence immunoassay (ECLIA).^4,5 Although these measurements can determine PCT in human serum with satisfied sensitivity and accuracy, they are usually costly and inconvenient, requiring time-consuming procedures, complicated equipment, and well-trained personnel; thus, these methods are not suitable for point-of-care testing (POCT). Therefore, the development of a rapid, sensitive and quantitative analytical tool to analyze PCT is highly desirable.

Immunochromatographic strip tests (IST), also called lateral flow tests or dipstick tests, combine chromatography with conventional immunoassay to offer a new analytical tool.^6,7 Because of its simplicity, rapidity and low cost in analysis of protein and other molecules,^8,9 IST attracts great research interests and has been widely used as an in-field and point-of-care diagnosis tool to detect and identify infectious diseases, cancer, cardio vascular problems, and biological warfare agents.^10,11 Colloidal gold nanoparticles are the most commonly employed reporters of IST;^11,12 however, this colloidal gold-based ISTs are generally qualitative or semi-quantitative for concentrated analytes.^13,14 Fluorescence-based assays attract more attention since they allows fast, sensitive, highly quantitative and multiplex detection of analytes of interest.^11,13,15 Currently, several types of fluorescent labels have been employed as IST reports, including fluorophores,^16,17 quantum dots (QDs),^18–20 up-converting phosphor nanoparticles (UCPs)^21,22 and dye-doped nanoparticles.^23–25 However, the most used fluorophores suffer from a narrow Stokes shift (20–30 nm), photo bleaching and low emission intensity, resulting in a decreased fluorescence intensity and limited sensitivity of the fluorescence labelling method.^12,26–28 The surface modification of stable and brighter fluorescent nanoparticles such as QDs and UCPs are difficult and laborious, which largely limits their large-scale application.^29–31 Therefore, it is highly desirable to find an accessible, bright and anti-photo bleaching label as a reporter of IST.

Thanks to the lanthanide chelates' specific properties of a wide Stokes shift, sharp emission profiles, bright fluorescence, excellent stability against photo-bleaching and long luminescence lifetime,^32–34 they have gained increasing attention as luminescent probes for sensing,^35,36 fluorescence microscopy bioimaging,^37–39 highly sensitive time-resolved fluoroimmunoassay (TR-FIA) etc.^33,40–42 The tris(dibenzoylmethane)mono(1,10-phenanthroline)europium(III) (Eu(DBM)₃phen) is one of the lanthanide chelates that has been proven to emit stable and bright fluorescence,^43–45 possessing potential as a probe for sensitive sandwich-type immunoassays.

Herein, we report, for the first time, the development of a fluorescence-based IST which employs Eu(DBM)₃phen-containing nanoparticles as reporters for the detection of PCT in human serum. We encapsulated Eu(DBM)₃phen into monodisperse polystyrene (PS) nanoparticles to provide a highly amplified signal. The carboxyl group modified on the PS particle surface could be easily modified with amino groups from the antibody to form a stable amide bond. Due to the advantages derived from EuPSNs and IST, a sensitive, specific and one-step strategy has been developed for PCT analysis in human serum. A homemade portable strip reader was fabricated to record fluorescence intensity on the test line and control line for POCT quantitative detection of PCT. Experimental results demonstrate that the developed IST has an excellent ability for rapid (15 min), sensitive (0.05 ng mL⁻¹) and quantitative analysis of PCT, and the assay results are comparable to which of the conventional ELISA.

Experimental

Chemicals and materials

Methylene benzoyl (DBM, 98%), 1,10-phenanthroline (phen, 97%), europium chloride(III)·hexahydrate (EuCl₃·6H₂O, 99.9%), styrene (st), acrylic acid (AA), dichloromethane, ethanol, potassium peroxydisulfate (KPS) and sodium hydroxide were purchased from Aladdin reagent (Shanghai, China). Sodium dodecyl sulfate (SDS), polyvinyl alcohol (PEG, avg. 20 000 M_w), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), bovine serum albumin (BSA),Tween-20, sodium casein, N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), procalcitonin (PCT) were purchased from Sigma (St. Louis, MO, USA). Nitrocellulose membrane (CN95) was purchased from Sartorius (Goettingen, Germany). Adsorption pad (SX42), sample pad (sb08) and conjugate pad (rb45) were purchased from Shanghai Jinbiao (Shanghai, China). Goat anti-rabbit IgG (GAR, >95%), rabbit IgG (RIgG, >95%) were purchased from Hangzhou Longji biotechnology Co., LTD (Hangzhou, China). Monoclonal mouse anti-human PCT antibody (MJG 05 and MJG 03, represented respectively with Ab1 and Ab2) was purchased from Hangzhou Qitai biotechnology Co., LTD (Hangzhou, China). All buffer solutions were prepared in laboratory. The water was prepared via the Milli-Q (Millipore, Milford, MA, USA). The other chemical reagents were of analytical grade and obtained through a standard commercial access.

Synthesis of Eu(DBM)₃phen

The Eu(DBM)₃phen was synthesized following the previous reports.^45,46 2 mmol phen (0.4 g), 6 mmol DBM (1.34 g), 6 mmol NaOH (0.24 g) and anhydrous ethanol (20 mL) were added to 100 mL round bottom flask under magnetic stirring and then heated at 50 °C in water bath. After 10 min, 2 mmol EuCl₃·6H₂O (0.52 g) dissolved in 20 mL ethanol was drop wisely added. Then 6 M NaOH was used to adjust the pH of the solution to 6–7. After stirring for 1 h, the precipitate was filtered off, washed with water and ethanol, dried at room temperature to yield Eu(DBM)₃phen as pale yellow solid.

Synthesis of carboxyl modified PS nanoparticles

The carboxylic PS nanoparticles were synthesized according to the previously reported literatures.^47,48 Typically, under gentle magnetic stirring, 5 mL of st and 20 μL of AA were added to 50 mL of deionized water in a 100 mL three-neck round bottom flask. The mixture was stirred for 15 min. Then 0.09 g of KPS in 2 mL deionized water was added, the mixture was deoxygenated by nitrogen bubbling at room temperature for 15 min. After degassing, the reaction mixture was gradually heated to 75 °C in an oil bath, and let react for 24 h. Then the reaction mixture was cooled to room temperature. The obtained latex spheres were washed three times with water and then dispersed in 0.25% (w/v) SDS aqueous solution at 3% (w/v).

Preparation of EuPSNs

The EuPSNs were synthesized via encapsulating Eu(DBM)₃phen into monodisperse PS nanoparticles. 100 mL suspension of carboxylic PS nanoparticles (3%, w/v, dispersed in 0.25% SDS aqueous solution) was added to a 200 mL Erlenmeyer flask, then 150 mg Eu(DBM)₃phen dissolved in 10 mL CH₂Cl₂ was then added and sonicated for 15 min to form a uniform suspension. The resulted suspension was magnetically stirred at room temperature for 24 h, followed by heating at 50 °C in water bath over night to completely evaporate the organic solvent CH₂Cl₂. The product was ultrasonically washed three times with ethanol and then three times with water by centrifugation. The resulted EuPSNs were then dispersed in water (1% w/v) and kept at 4 °C as stock.

Preparation of antibody-labelled EuPSNs

The EuPSNs were functionalized with anti-PCT capture antibody (Ab2) according to literature with little modification.^49,50 Briefly, EuPSNs (1%, w/v) were washed twice with water via centrifugation, and then dispersed in MES (10 mM, pH = 5.5) by sonication. EDC (5 mg mL⁻¹) and sulfo-NHS (5 mg mL⁻¹) were added to activate the carboxylic acid groups on the surface for 30 min. After that, the activated particles were washed with water and then dispersed in MES (10 mM, pH = 6.2, 0.05% v/v Tween20). 0.2 mg mL⁻¹ Ab2 (the concentration of Ab2 was optimized in Fig. S-1A†) was then added and incubated at 25 °C for 2 h with slow rotation and kept at 4 °C overnight. The Ab2 labeled EuPSNs (EuPSNs-Ab2) were harvested by centrifugation, followed by being ultrasonically redispersed in 20 mM PBS (0.5% wt casein, 2.5% wt BSA, 1% wt saccharose, 2% wt PEG-2000 and 0.03 wt% NaN₃, pH = 8.0) to prepare diluted dispersion (0.1% w/v, EuPSNs-Ab2) and kept at 4 °C. The preparation of GAR labeled EuPSNs (EuPSNs-GAR, 0.1% w/v) was similar to this method.

Fabrication of immunochromatographic strip

The immunochromatographic strip was composed of five components, a plastic backing, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad. The sample pads and the conjugate pads were treated with 20 mM phosphate buffer containing 1% wt BSA, 2.5% wt sucrose, 1% v/v Tween20, 0.3% wt PVP K30, and 0.02% wt sodium azide (pH 7.4) and dried at 37 °C and 25% relative humidity overnight. The Ab1 (1 mg mL⁻¹) (the concentration of Ab1 was optimized in Fig. S-1B†) or the RIgG (1 mg mL⁻¹) in PBS (1% wt trehalose) was dispensed at the test or the control line on the nitrocellulose membrane, using a HM3030XYZ dispenser (Shanghai KinbioTech Co., Ltd., China) at a rate of 0.9 μL cm⁻¹ and a speed of 4 cm s⁻¹ and then dried at 37 °C and 25% relative humidity overnight. The antibodies-labeled EuPSNs dispersion (EuPSNs-Ab2 and EuPSNs-GAR were mixed at the ratios of the volume 10 : 1) was applied to the treated conjugate pad at a rate of 10 μL cm⁻¹ and then lyophilized completely. The absorption pad, nitrocellulose membrane, pretreated conjugate pad, and sample pad were attached to a plastic backing and assembled as a strip with a 1 to 2 mm overlap, sequentially. The assembled plate was cut into 3 mm-wide pieces, using an automatic strip cutter ZQ2000 (Shanghai Kinbio Tech Co., Ltd., China). The generated strip products were packaged in a plastic bag with desiccant and stored at RT.

Design and fabrication of fluorescence immunoassay reader

To read the fluorescence, a portable, compact, easy-to-use reader with low cost was developed, which incorporated an optical module, a linear stage, and an electrical module with a microcontrollers. The optical module can be split into two parts: excitation and detection. For excitation, a 365 nm ultraviolet diode with a viewing angle of 15° (HaSun Optoelectronics Co., Limited, HK, China) was employed as a light source. The emitted light was collimated with a collimating lens. The collimated light was reflected perpendicularly by a dichroic mirror, and then focused on the IST strips by a glass lens, thereby forming a circular shape of excitation light with a diameter of ∼1 mm. For detection, the emitted fluorescence light was collimated and passed through the dichroic mirror. After being filtered by a band-pass filter (600–630 nm, Giai Photonics Co., Ltd, Shenzhen), the fluorescence was focused by a second lens on a silicon photodiode detector (PIN-13DSB, OSI Optoelectronics). The generated photocurrent by the photodiode was converted into a voltage (I/V) by a rail-to-rail low power FET-input Op amplifier (AD822, National Semiconductor), and amplified by the same Op amplifier with again of 100. This amplified signal was recorded though a 12 bit A/D converter and analyzed by the ARM microcontroller (STM32F407ZET6, STMicroelectronics). A compact linear stage was constructed to facilitate smooth cassette movement with high resolution for strip scanning. After each scanning, a smooth curve with two peaks corresponding to the fluorescence emitted from T and C lines was obtained. By integrating the two peaks respectively, the areas of S_T and S_C were calculated. The ratio of area (S_T/S_C) was employed to calibrate versus PCT concentration of sample. The optical reader was accessed with a touch-screen interface, and the test results could be saved and printed.

Quantitative detection of fluorescent IST

75 μL of bovine serum containing varied amount of PCT (0.05, 0.2, 1, 5, 10, 20, 50 ng mL⁻¹) was added on the sample pad of an IST strip. The bovine serum without PCT was used as control. Both the samples and control were migrated toward the absorption pad by capillary action. After 15 min, the test strip was analyzed by the designed fluorescence immunoassay reader. The ratio of integrated area (S_T/S_C) was acquired and shown in the touch screen. Every sample was assayed in triplicate. A standard curve used for quantification was obtained by calibrating the ratio of area S_T/S_C versus PCT concentration of the samples. For clinical experiments, 75 μL of human serum was added onto the sample pad. The results were obtained by reading the optical response with fluorescence immunoassay reader after 15 min. All measurements were conducted at RT.

ELISA detection of PCT

Each well of the microtiter plates (Corning Costar® 96-Well EIA/RIA Stripwell™ Plates) was coated with 100 μL of Ab1 at concentration of 1.0 μg mL⁻¹ and incubated over night at 4 °C. Unbound coating antibody was removed from the plate with a washing buffer (0.1% wt BSA, 0.05% v/v Tween-20, pH = 7.4), and each well was blocked with 1% wt BSA in PBS at 37 °C for 1 h. 100 μL of PCT samples with different concentration or standard samples were added in 96-well plate for incubating 1 h. Then, each well was washed with 250 μL washing buffer for four times. 100 μL of Ab2-HRP (2.0 μg mL⁻¹) conjugate solution was injected into each well and incubated for 1 h. After each well was washed with 250 μL washing buffer for four times, 100 μL TMB solution was then added to each well and incubated for 15 min at room temperature. The reaction was terminated by adding 50 μL of 2 M H₂SO₄ to each well. The absorbance was measured at 450 nm with a Multiskan™ FC Microplate Photometer (Thermo Scientific).

Characterization

The surface profile characteristic of particles was studied using SEM (JCM-6000). UV-visible spectrum performed on a Shimadzu spectrophotometer (UV-2600). The fluorescence spectrum was obtained by fluorescence spectrophotometer of Shimadzu Ltd. (RF-5301PC). The size of particles and the process of antibody labeling were characterized by the particle size characterization instrument of Malvern Instruments Ltd. (Zetasizer Nano ZSP).

Results and discussion

Preparation of EuPSNs

To evaluate the superiority of a lanthanide chelate as a IST reporter, there are five major technical aspects to be considered including: (1) water solubility or good dispensability in water; (2) formation of stable complex with a high thermodynamic stability and kinetic inertness; (3) a high photochemical stability and quantum yield; (4) a reactive group allowing covalent attachment to biomolecules; (5) maintenance of high biological activity of chelate label-conjugated biomolecules after labeling. Despite thorough research, these properties are rarely all present in a single chelate. Thus encapsulating lanthanide chelates into water-disperse spheres are preferred. In previous studies, bright lanthanide chelates-doped silica microspheres were fabricated by encapsulating lanthanide chelates into silica.²³ We here prepared EuPSNs by encapsulating Eu(DBM)₃phen into carboxyl-modified PS nanoparticles. EuPSNs were obtained though swelling prepared PS nanoparticles with a mixture of Eu(DBM)₃phen and CH₂Cl₂ in microemulsion, followed by completely evaporating the organic solvent CH₂Cl₂, leaving Eu(DBM)₃phen precipitated within the hydrophobic PS nanoparticles (Fig. 1A). The PS nanoparticles were highly monodisperse with a diameter of 237 nm (Fig. 1B). After encapsulation of Eu(DBM)₃phen, no apparent changes of surface topography could been observed (Fig. 1C). The resulted EuPSNs were still very stable in water and no apparent aggregation or sediment was observed after standing for 3 days at RT (Fig. S-3†). The absorption and emission spectra of the EuPSNs excited at 365 nm were presented in Fig. 1D and E with a half-width peak of about 10 nm. EuPSNs emitted strong fluorescence at 613 nm under UV excitation as shown in Fig. 1E. To determine the number of lanthanide chelates per particle, a certain amount of dried EuPSNs was dissolved in CH₂Cl₂ to prepare a homogeneous solvent. The number of dried particle dissolved was determined by using the weight of one single particle (4/3πr³ρ) divided by that of all the particles in the solution.^33,51,52 The number of lanthanide chelates in the solvent was then calculated by measuring the fluorescence signal against a lanthanide calibrator in the same solvent. The number of lanthanide chelates per particle was determined by dividing the number of lanthanide chelates by the number of EuPSNs dissolved in the solvent. Each PS particle contained ∼142 000 Eu(DBM)₃phen molecules, which provided a highly amplified signal for fluorescence-based IST.

Fig. 1 Preparation and characterization of EuPSNs. Schematic preparation of EuPSNs (A). The SEM images of the PS nanoparticles before (B) and after (C) encapsulation of Eu(DBM)₃phen. The absorption (D) and emission (E) spectra of the EuPSNs. The inset in (E) shows photograph of EuPSNs under natural light and UV light.

Antibody conjugation

After activation by EDC and sulfo-NHS, the antibodies were directly conjugated to EuPSNs and formed a stable amide bond. Previous studies reported that the proper conjugation of antibodies is crucial for the efficiency of IST strips and the aggregation of the labeled nanoparticles was the leading cause of conjugation failure.⁵³ To solve this problem, Tween20 (0.05% w/v) was added in the conjugate solution to stabilize the coupled EuPSNs and the coupling process was monitored by DLS analysis. Fig. 2 showed the hydrodynamic diameter of EuPSNs increased from 244 nm to 268 nm after coupling, suggesting that the conjugation of EuPSNs and antibodies happened. The narrow width and symmetry of the scattering graph indicated that the bare EuPSNs and EuPSNs-Ab2 conjugate were highly monodisperse and no aggregation occurred during the conjugation process (Fig. 2). Notably, the polydispersity index (PDI) of the conjugates showed the prepared conjugates were highly stable and only had minimal changes after 20 h in PBS (PDI = 0.010, 0.010 and 0.012 after standing for 0, 10 and 20 h respectively, Fig. 2B). The fluorescence property of EuPSNs-Ab2 also showed very little change in water, 20 mM PBS and 100 mM PBS respectively (Fig. S-4†).

Fig. 2 DLS analysis of bare EuPSNs (A) and (B) Ab2-EuPSNs conjugate standing for 0, 10 and 20 h in PBS.

Principle of EuPSNs-based IST

Fig. 3 showed a schematic diagram of detection principles for the PCT and the apparatus employed for IST. Fig. 3A showed the fabrication process of EuPSNs based IST probes. The capture antibodies Ab2 and GAR were chemically conjugated to EuPSNs via the classic EDC/NHS reaction. Fig. 3B showed the principles for PCT detection and construction of IST strips. Two types of conjugates, EuPSNs-Ab2 and EuPSNs-GAR, were dispensed onto the conjugate pad and dried there. Upon addition of liquid samples onto the adjacent sample pad, the conjugates were rehydrated and consequently released into the migrating fluidic serum. The EuPSNs-Ab2 was able to recognize and link to the antigens. Driven by capillary force, the mixture of the fluids (i.e., two types of conjugates and potential PCT antigens) then migrated across both the T-line and the C-line, where Ab1 (PCT antibodies) and RIgG were pre-immobilized, respectively. Subsequently, agglutination of conjugates released from the conjugate pad, such as the triplet constructs of EuPSNs-Ab2, PCT antigens and Ab1 in a sandwich format, will be captured in the “T-line” on the NC membrane. In contrast, EuPSNs-GAR conjugates are recognized by RIgG and immobilized in the C-line, an internal control that reflects the effective release of EuPSNs conjugates from the conjugate pad thereby.

Fig. 3 Principles of quantitative detection of PCT by EuPSNs-based IST strip. (A) A schematic diagram of the EuPSNs-antibodies probes. (B) The principles for PCT detection and construction of IST strip. (C) Schematic of the optical system of the designed IST reader and (D) the obtained scanning curve by the IST reader, and the inset shows the photograph of the designed IST reader.

To quantify the fluorescence generated from the IST strips, we designed and fabricated a compact, portable, and cost-friendly fluorescence reader. Fig. 3C showed the schematic of the optical system of the designed IST reader. Due to the wide Stokes shift (365 nm excited and 613 nm emitted) and bright fluorescence of EuPSNs, a low-cost and stable 365 nm ultraviolet diode, instead of traditional expensive mercury lamps and lasers, was employed as an exciting light source without coupling filter. As for the collection of fluorescence signal, the most popular detectors used for traditional fluorescence detection systems are photo multiplier tubes (PMTs), avalanche photodiodes (APDs), and photon counting modules (PCMs). However, such systems are complex, bulky, and costly, which are not compatible with the superiorities of POCT.³⁸ To make a miniaturized and cost effective IST fluorescence reader, we here employed a silicon photo diode detector by which the emitted fluorescence from the IST strip was recorded after filtered by a band-pass filter. A step motor was used to facilitate the smooth movement of strip with high resolution for fluorescence scanning. After the focused spot of the excited light scanned through the strip, a smooth curve with two peaks corresponding to the fluorescence emitted from T and C lines was obtained (Fig. 3D). The inset in Fig. 3D showed the photograph of the real IST reader. It was compact (L × W × H, 30 cm × 20 cm × 12 cm), light (0.75 kG), user-friendly and cost effective.

Quantitative detection of EuPSNs-based IST

Thanks to the satisfying signal brightness of EuPSNs, EuPSNs-based IST allowed both qualitative and quantitative analysis. Qualitative control of the PCT levels in new-born calf serums using naked eye visual detection was first examined. Under the excitation of a high-power 365 nm LED array, a qualitative estimation of PCT concentration could be easily judged by the fluorescence strength. As can been seen in Fig. 4A, the fluorescence brightness of the T lines was observed to proportionally change with the concentration of PCT (0, 0.05, 0.2, 1, 5, 10, 20, 50 ng mL⁻¹). In order to obtain quantitative results, the fluorescence signal on the strips was read by our designed IST reader. Fig. 4B showed the recorded curves from samples as in Fig. 4A. Previous studies suggested that application of the fluorescence signal ratio (T/C) could minimize the strip-to-strip effects in the immunoassay detection as well as shorten the strip interpretation time.^51,54 Here, we calculated the areas of S_T and S_C by integrating the two peaks respectively after each scanning. The ratio of areas (S_T/S_C) was employed to calibrate versus PCT concentration of samples. It was found that S_T/S_C got stable 15 min after adding the sample(see Fig. S-2†), so the interpretation time of the developed strip was determined to be 15 min. Fig. 4C showed the four parameters logistic curve of the S_T/S_C ratios against PCT concentrations. Error bars were based on three duplicate measurements at different concentrations. The regression equation was y = A₂ + (A₁ − A₂)/(1 + (x/x₀)^p) (A₂ = 33.46, A₁ = 0.6757, x₀ = 12.12, p = 1.060, where x represented PCT concentration, y represented S_T/S_C) with a reliable correlation coefficient of R² = 0.993. As the detection result shown, the limit of detection (LOD) of this method was 0.05 ng mL⁻¹ and the detection sensitivity was 0.04 ng mL⁻¹ PCT (the result was calculated plus three times the standard deviation at the nose of 0 ng mL⁻¹ PCT sample detection).

Fig. 4 (A) Qualitative analysis of the PCT levels (0, 0.05, 0.2, 1, 5, 10, 20, 50 ng mL⁻¹ respectively) in new-born calf serums. (B) The scanning curves of the EuPSNs-based IST strips in (A). (C) The standard curve of PCT detection using EuPSNs-based IST.

The life-time stability of the EuPSNs-based IST biosensor was studied by using strips stored at RT for varied time to detect a spiked PCT sample (5 ng mL⁻¹ PCT in new-born calf serums). Fig. 5 indicates that the obtained signal S_T/S_C during six month has only minor change (<10%) compared with which obtained by the freshly prepared strips. These results show the fabricated biosensor owns a life time of six months at least at RT. The reproducibility and recovery were validated by assaying 0.5, 2.5, and 10 ng mL⁻¹ PCT standard examples, using identical batches of EuPSNs-Ab2 and EuPSNs-GAR. The obtained results showed that the coefficients of variation (CVs, n = 5) of the assay were 10.3%, 6.7% and 4.2% for 0.5, 2.5, and 10 ng mL⁻¹ PCT, and the recoveries ranged from 86.6% to 114.5%. The improved reproducibility and recovery were possibly due to the preparation of monodisperse antibodies-conjugated EuPSNs and the proposed quantitative calibration.

Fig. 5 The life-time stability of the strips. Strips stored at RT for varied time were employed for detection of a spiked PCT sample (5 ng mL⁻¹ PCT in new-born calf serums) in triplet.

Determination of PCT in human serum

To evaluate the application of EuPSNs-based IST in clinical diagnosis, 20 different PCT concentration samples were obtained from Shenzhen People's Hospital according to the request of the local medical and health community. Before detection, the samples were centrifuged at 2000 rpm and 4 °C for 10 min to precipitate the blood erythrocytes. The supernatants were used as the detection samples. The acceptance of the new EuPSNs-based IST for PCT was compared with a traditional ELISA method by blindly analyzing the obtained human serum samples (PCT concentrations from 0.15 to 4.75 ng mL⁻¹). The results (Fig. 6) show that the two methods exhibited good agreement with a highly significant correlation (R² = 0.98). The slope and intercept of the regression equation between two methods were close to 1 and 0 (1.05 and 0.011), respectively, revealing a good agreement between the two analytical methods. However, the proposed EuPSNs-based IST took only 15 min to complete one sample analysis, which was much shorter than the time the traditional ELISA took (∼120 min).

Fig. 6 Correlation analysis of the detectable concentration between EuPSNs-based IST and traditional ELISA in 20 human serum samples with PCT concentrations from 0.15 to 4.75 ng mL⁻¹.

Conclusions

In conclusion, a sensitive, rapid, low cost, and user-friendly EuPSNs-based IST sensor was successfully developed, using Eu(DBM)₃phen-encapsulated PS nanoparticles as fluorescent reporters to assay PCT in human serum samples. In order to make the PCT biosensor applicable for point-of-care monitoring, a portable and cost-friendly fluorescence strip reader was designed and fabricated. The signal curve from EuPSNs-based IST sensor were recorded and processed as ratio of integrated area (S_T/S_C) to minimize signal variations from strip to strip and sample variability with the designed reader. Using the developed biosensor system combining the EuPSNs-based IST strip and the fluorescence reader, we could obtain a LOD of 0.05 ng mL⁻¹ and a sensitivity of 0.04 ng mL⁻¹ PCT. The reliability of EuPSNs-based IST sensor for PCT analysis was invalidated by a traditional ELISA method, the obtained results demonstrated a good correlation. What more, the time for one sample detection by the EuPSNs-based IST sensor only needs 15 min, which is reduced by 8-fold compared to the traditional ELISA (∼120 min). These results suggest that the developed assay could be used for point-of-care test of PCT in serum.

Acknowledgements

This work is supported by National Natural Science Foundation of China (Grant 21102007, 21372023, 21374064), MOST 2015DFA31590, the Shenzhen Science and Technology Innovation Committee (SW201110018, SGLH20120928095602764, ZDSY20130331145112855, JSGG20140519105550503, JSGG20160301095829250) and the Shenzhen Peacock Program (KQTD201103).

References

1. S. Agarwal, N. Akbas, E. P. Soundar, G. Gonzalez and S. Devaraj, Clin. Biochem., 2015, 48, 886–890.
2. R. Pierce, M. T. Bigham and J. S. J. Giuliano, Curr. Opin. Pediatr., 2014, 26, 292–298.
3. A. A. H. Zeitoun, S. S. Gad, F. M. Attia, A. S. Abu Maziad and E. F. Bell, Scand. J. Infect. Dis., 2010, 42, 299–305.
4. P. Schuetz, M. Christ-Crain, A. R. Huber and B. Müller, Clin. Biochem., 2010, 43, 341–344.
5. Y. Sakr, C. Sponholz, F. Tuche, F. Brunkhorst and K. Reinhart, Infection, 2008, 36, 396–407.
6. G. Liu, Y.-Y. Lin, J. Wang, H. Wu, C. M. Wai and Y. Lin, Anal. Chem., 2007, 79, 7644–7653.
7. H. Nian, J. Wang, H. Wu, J.-G. Lo, K.-H. Chiu, J. G. Pounds and Y. Lin, Anal. Chim. Acta, 2012, 713, 50–55.
8. Y. Jin, J.-W. Jang, C.-H. Han and M.-H. Lee, J. Agric. Food Chem., 2005, 53, 7639–7643.
9. Y.-Y. Lin, J. Wang, G. Liu, H. Wu, C. M. Wai and Y. Lin, Biosens. Bioelectron., 2008, 23, 1659–1665.
10. G. M. Weller, Fresenius' J. Anal. Chem., 2000, 366, 635–645.
11. X. Huang, Z. P. Aguilar, H. Xu, W. Lai and Y. Xiong, Biosens. Bioelectron., 2016, 75, 166–180.
12. M. Sajid, A.-N. Kawde and M. Daud, J. Saudi Chem. Soc., 2015, 19, 689–705.
13. Q. Yang, X. Gong, T. Song, J. Yang, S. Zhu, Y. Li, Y. Cui, Y. Li, B. Zhang and J. Chang, Biosens. Bioelectron., 2011, 30, 145–150.
14. J. P. Golden, J. S. Kim, J. S. Erickson, L. R. Hilliard, P. B. Howell, G. P. Anderson, M. Nasir and F. S. Ligler, Lab Chip, 2009, 9, 1942–1950.
15. K. Boldt, O. T. Bruns, N. Gaponik and A. Eychmüller, J. Phys. Chem. B, 2006, 110, 1959–1963.
16. N. Khreich, P. Lamourette, H. Boutal, K. Devilliers, C. Créminon and H. Volland, Anal. Biochem., 2008, 377, 182–188.
17. J. S. Ahn, S. Choi, S. H. Jang, H. J. Chang, J. H. Kim, K. B. Nahm, S. W. Oh and E. Y. Choi, Clin. Chim. Acta, 2003, 332, 51–59.
18. H. Qu, Y. Zhang, B. Qu, H. Kong, G. Qin, S. Liu, J. Cheng, Q. Wang and Y. Zhao, Biosens. Bioelectron., 2016, 81, 358–362.
19. C. Wang, F. Hou and Y. Ma, Biosens. Bioelectron., 2015, 68, 156–162.
20. X. Li, W. Li, Q. Yang, X. Gong, W. Guo, C. Dong, J. Liu, L. Xuan and J. Chang, ACS Appl. Mater. Interfaces, 2014, 6, 6406–6414.
21. R. S. Niedbala, H. Feindt, K. Kardos, T. Vail, J. Burton, B. Bielska, S. Li, D. Milunic, P. Bourdelle and R. Vallejo, Anal. Biochem., 2001, 293, 22–30.
22. L. Li, L. Zhou, Y. Yu, Z. Zhu, C. Lin, C. Lu and R. Yang, Diagn. Microbiol. Infect. Dis., 2009, 63, 165–172.
23. F. Zhang, M. Zou, Y. Chen, J. Li, Y. Wang, X. Qi and Q. Xue, Biosens. Bioelectron., 2014, 51, 29–35.
24. L.-W. Song, Y.-B. Wang, L.-L. Fang, Y. Wu, L. Yang, J.-Y. Chen, S.-X. Ge, J. Zhang, Y.-Z. Xiong, X.-M. Deng, X.-P. Min, J. Zhang, P.-J. Chen, Q. Yuan and N.-S. Xia, Anal. Chem., 2015, 87, 5173–5180.
25. Z. Wang, H. Li, C. Li, Q. Yu, J. Shen and S. De Saeger, J. Agric. Food Chem., 2014, 62, 6294–6298.
26. G. A. Posthuma-Trumpie, J. Korf and A. van Amerongen, Anal. Bioanal. Chem., 2009, 393, 569–582.
27. X. Gao, L.-P. Xu, S.-F. Zhou, G. Liu and X. Zhang, Am. J. Biomed. Sci., 2014, 6, 41–57.
28. Z. Li, Y. Wang, J. Wang, Z. Tang, J. G. Pounds and Y. Lin, Anal. Chem., 2010, 82, 7008–7014.
29. L. Huang, Z. Luo and H. Han, Chem. Commun., 2012, 48, 6145–6147.
30. S. Wu, N. Duan, X. Ma, Y. Xia, H. Wang, Z. Wang and Q. Zhang, Anal. Chem., 2012, 84, 6263–6270.
31. F. Wang and X. Liu, Chem. Soc. Rev., 2009, 38, 976–989.
32. M.-L. Järvenpää, K. Kuningas, I. Niemi, P. Hedberg, N. Ristiniemi, K. Pettersson and T. Lövgren, Clin. Chim. Acta, 2012, 414, 70–75.
33. H. Härmä, T. Soukka and T. Lövgren, Clin. Chem., 2001, 47, 561–568.
34. O. L. Malta, H. F. Brito, J. F. S. Menezes, F. R. Gonçalves e Silva, C. de Mello Donegá and S. Jr Alves, Chem. Phys. Lett., 1998, 282, 233–238.
35. C.-F. Chow, H.-K. Kong, S.-W. Leung, B. K. W. Chiu, C.-K. Koo, E. N. Y. Lei, M. H. W. Lam, W.-T. Wong and W.-Y. Wong, Anal. Chem., 2011, 83, 289–296.
36. J. Hu, X. Jiang, L. Wu, K. Xu, X. Hou and Y. Lv, Anal. Chem., 2011, 83, 6552–6558.
37. D. Jin and J. A. Piper, Anal. Chem., 2011, 83, 2294–2300.
38. L. Novak, P. Neuzil, J. Pipper, Y. Zhang and S. Lee, Lab Chip, 2007, 7, 27–29.
39. K. Hanaoka, K. Kikuchi, S. Kobayashi and T. Nagano, J. Am. Chem. Soc., 2007, 129, 13502–13509.
40. W. Lei, A. Dürkop, Z. Lin, M. Wu and S. O. Wolfbeis, Microchim. Acta, 2003, 143, 269–274.
41. A. K. Hagan and T. Zuchner, Anal. Bioanal. Chem., 2011, 400, 2847–2864.
42. A. Scorilas, A. Bjartell, H. Lilja, C. Moller and E. P. Diamandis, Clin. Chem., 2000, 46, 1450–1455.
43. H. Liang, B. Chen, Q. J. Zhang, Z. Q. Zheng, H. Ming and F. Q. Guo, J. Appl. Polym. Sci., 2005, 98, 912–916.
44. A. K. Singh, S. K. Singh, H. Mishra, R. Prakash and S. B. Rai, J. Phys. Chem. B, 2010, 114, 13042–13051.
45. E. Moretti, A. Talon, L. Storaro, A. Le Donne, S. Binetti, A. Benedetti and S. Polizzi, J. Lumin., 2014, 146, 178–185.
46. B. Yan, H.-j Zhang, S.-b Wang and J.-z Ni, Mater. Chem. Phys., 1997, 51, 92–96.
47. B.-Z. Yang, L.-W. Chen and W.-Y. Chiu, Polym. J., 1997, 29, 737–743.
48. X. Du and J. H. He, J. Appl. Polym. Sci., 2008, 108, 1755–1760.
49. R.-Q. Zhang, S.-L. Liu, W. Zhao, W.-P. Zhang, X. Yu, Y. Li, A.-J. Li, D.-W. Pang and Z.-L. Zhang, Anal. Chem., 2013, 85, 2645–2651.
50. T. Liao, F. Yuan, H. Yu and Z. Li, Anal. Methods, 2016, 8, 1577–1585.
51. X. Huang, Z. P. Aguilar, H. Li, W. Lai, H. Wei, H. Xu and Y. Xiong, Anal. Chem., 2013, 85, 5120–5128.
52. P. Huhtinen, M. Kivelä, O. Kuronen, V. Hagren, H. Takalo, H. Tenhu, T. Lövgren and H. Härmä, Anal. Chem., 2005, 77, 2643–2648.
53. T. O. Harasym, P. Tardi, S. A. Longman, S. M. Ansell, M. B. Bally, P. R. Cullis and L. S. L. Choi, Bioconjugate Chem., 1995, 6, 187–194.
54. Y. Gu, Y. Yang, J. Zhang, S. Ge, Z. Tang and X. Qiu, Instrum. Sci. Technol., 2014, 42, 635–645.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra23816e

‡ These authors contributed equally to the manuscript.

---