A. Chemicals and reagents
Free cortisol, bovine serum albumin (BSA), cortisol-BSA, and surfactant 10G (S10G) were purchased from Fitzgerald Industries International (Acton, MA, USA). The anti-cortisol antibody was purchased from Abcam (Cambridge, UK), and the nitrocellulose (NC) membrane (FF80HP) was purchased from GE Healthcare (Chicago, IL, USA). Salivette© Cortisol, which was used to collect saliva samples, was purchased from Sarstedt (Numbrecht, Germany). The absorbent pad (Grade222), conjugate pad (6613), signal amplification pad (8964), and sample pads (Grade222) were purchased from Ahlstrom-Munksjö (Helsinki, Finland). The NC membrane without backing (HF180UBXSS) was purchased from EMD Millipore (Burlington, MA, USA), and the Vivid plasma separation-GX membrane (T9EXPPA0200S00X) and asymmetric membrane - MMM0.45 (T9PA045W000M) were purchased from Pall Corporation (Port Washington, NY, USA). PVA tape (126057) and a general double-sided tape (DTS-310) were purchased from Buho, Inc. (Daegu, Korea) and DUCKSUNG HITECH, Inc. (Gwangju, Korea), respectively. Gold nanoparticles (EM.GC15) and the TMB substrate reagent set (555214) were purchased from BB International (Cardiff, UK) and BD Biosciences (San Jose, CA, USA), respectively. The ELISA microplate (655061) was purchased from Greiner Bio-One International GmbH (Mühlkreis, Austria). Phosphate-buffered saline (PBS), sodium phosphate (monobasic), and sodium phosphate (dibasic) were purchased from Biosesang Co. (Sungnam, Korea). Protein saver was purchased from TOYOBO Co., Ltd. (Osaka, Japan). An anti-mouse IgG antibody produced in goat, boric acid, sodium tetraborate decahydrate, trisodium citrate dihydrate, citric acid, polyvinylpyrrolidone (10K) (PVP 10K), D-(+)-trehalose dehydrate, Triton™ X-100, Tween 20, dimethyl sulfoxide (DMSO), gold(III) chloride trihydrate, hydroxylamine hydrochloride, sulfuric acid, cortisone, corticosterone (CORT), progesterone (P4), 17α-hydroxyprogesterone (17-OHP4), human serum, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Normal human saliva fluid and normal human urine fluid were purchased from MyBioSource (San Diego, CA, USA). Water was purified using a lab water purification system (PURELAB® Option-Q, ELGA LabWater Ltd., Lane End, UK).
B. Characterization of PVA tape
The structure of PVA tape was characterized by field emission scanning electron microscopy and light microscopy. The PVA tape characteristics were also compared to a general double-sided tape to determine its delayed-release effect. AuNP colloid (15 nm in diameter, 1× AuNP, λmax O.D. = 1.0) was immobilized on an NC membrane (1 μL·cm-1), and then gold(III) chloride trihydrate (0.1 M) was applied on the signal amplification pad. The general double-sided and PVA tapes were attached beneath the signal amplification pad. The sample solution, 10% (v/v) DMSO/PBS, and signal amplification solution, 10 mM hydroxylamine in 10 mM citrate (pH 3.8), were applied onto the sample and signal amplification pads, respectively. The weight of the PVA tape was measured at five time points after loading the buffer. The PVA and general double-sided tapes (size, 3.8 × 8.0 mm2) were placed in an e-tube containing citrate buffer (10 mM, pH 3.8) and vigorously vortexed. After 15 min, the tapes were dried completely, and their weights were measured using an ultra-microbalance.
Effective delayed release from the PVA tape was confirmed as described above with different membranes. To determine whether the PVA tape impacts the immunoassay, anti-mouse IgG antibodies (1 mg·mL-1) were immobilized on an NC membrane (1 μL·cm-1), and then AuNP–antibody conjugate (three-fold) was applied on a conjugate pad. The PVA tape was attached beneath the signal amplification pad, and the sample and signal amplification solutions were applied to the sample and signal amplification pads, respectively.
C. Preparation of AuNP conjugate
The anti-cortisol antibody (5 μL, 2 mg·mL-1) was added to a mixture of 1 mL of AuNP colloid (15 nm in diameter, 1× AuNP, λmax O.D. = 1.0) and 100 μL of 0.1 M borate buffer (pH 8.5). After incubation for 30 min at 21°C, 10 µL of protein saver (100 mg·mL-1 in PBS) was added to the mixture to block non-specific binding sites on the AuNP surface. After incubation for 1 h at 21°C, the mixture was centrifuged at 15 602 × g for 15 min at 10°C. The supernatant was discarded and the AuNP conjugate was resuspended in 1 mL of 10 mM borate buffer (pH 8.5), and the AuNP conjugate was concentrated by 10-fold and stored in storage buffer composed of 5% (w/v) D-(+)-trehalose, 0.5% (w/v) Protein saver, 0.2% (v/v) Tween 20, and 1% (v/v) Triton™ X-100 in PBS, at 4°C until use.
D. Preparation of α-trapLFI sensor using PVA tape
To prepare an α-trapLFI sensor, 250 μg·mL-1 cortisol–BSA and 1 mg·mL-1 anti-mouse IgG antibody were immobilized on an NC membrane (1 μL·cm-1) to create two deletion zones and one detection zone, respectively; the distance between each zone was approximately 2.5 mm. An absorbent pad was attached on top of the membrane with a 2-mm overlap. The AuNP conjugate was diluted by 1000-fold with a solution containing 1% (w/v) PVP 10K and 0.5% (w/v) S10G in PBS, and 3.61 μL of diluted AuNP conjugate was applied to the conjugate pad. Next, 8.2 μL of solution containing 0.1 M gold(III) chloride trihydrate in 10 mM phosphate buffer (pH 7.4) was applied to the signal amplification pad
The AuNP conjugate-loaded conjugate pad was attached to the bottom of the prepared strip, and a sample pad was placed underneath it to load the sample. The PVA tape was attached to the membrane at 2-mm above the conjugate pad, and a gold(III) chloride trihydrate-loaded signal amplification pad was placed in the middle of the PVA tape. The assembled membrane was cut into 3.8-mm wide strips and stored in a humidity-controlled chamber (21°C; 23% relative humidity) until use. Before use, the strip was placed in a plastic case provided by Infopia Co., Ltd. (Anyang, Gyeonggi, Korea).
E. Cortisol analysis in buffer, saliva, serum, and urine samples
Cortisol solutions at various concentrations in DMSO, human saliva, serum, and urine were diluted 10-fold in 1× PBS buffer (10% concentration). The signal amplification solutions were prepared at 10 mM in citrate buffer (pH 3.8). Subsequently, 100 μL of the prepared sample solution and 20 μL of the signal amplification solution were applied to the sample and signal amplification pads of the strip, respectively. The strip was then incubated at 21°C for 20 min. In addition, several other steroid hormones were also analyzed with same methods described above. All images were obtained using a ChemiDoc XPS+ imaging system (Bio-Rad, Hercules, CA, USA).
F. Collection of saliva samples
Human saliva samples were obtained from 20 volunteers from the Gwangju Institute of Science and Technology (GIST) in Gwangju, Korea. We collected samples of saliva from healthy individuals between 08:00 and 09:00 using a saliva collecting kit. The participants were asked to refrain from eating, drinking, and performing oral hygiene procedures until the morning of saliva collection. All participants provided informed consent to participate in the study. The study was approved by GIST (Approval number: 20190510-BR-45-08-02). The collected saliva samples were centrifuged at 1274 × g for 5 min to remove cell debris and other proteins, and the collected saliva samples were immediately used for testing. All experiments were performed in accordance with relevant guidelines and regulations.
G. Analysis of cortisol in human saliva samples
The results obtained using the sensor platform were compared to those obtained with an enzyme-linked immunosorbent assay (ELISA). Briefly, ELISA was performed in 96-well microplates. The anti-cortisol antibody (100 μL, 4 μg·mL-1) was added to each well, and the plates were incubated at 4°C overnight. After removing unbound antibodies by washing the wells with PBS-Tween (137 mM NaCl, 0.05% (v/v) Tween 20, pH 7.4), the wells were blocked with 200 μL of BSA solution (0.5 mg·mL-1 in PBS) at 21°C for 2 h. Next, individual wells were incubated with 100 μL of the diluted saliva samples and cortisol-HRP conjugate (1 μg·mL-1) mixture at 21°C for 5 h. After removing all unbound proteins, the samples were incubated with TMB substrate solution (50 μL) in the dark at 21°C for 10 min. Finally, sulfuric acid (50 μL, 2 N) was added to immediately stop the enzyme reaction. The absorbance of the solution was determined at 450 nm using a spectrometry system (Cytation 5; BioTek Instruments, Winooski, VT, USA).
H. Instruments
A microcentrifuge (smart R17 plus; Hanil Scientific Inc., Gimpo, Korea) was used to separate the AuNP conjugate, and a multi-purpose centrifuge (1580R; LABOGENE Co., Ltd., Lillerød, Denmark) was used to collect saliva samples. A drying oven (KO-100; LK Lab Co., Namyangju, Korea) was used to dry the membranes and all pads following antibody and sample loading. A dispenser (DCI100; Zeta Co., Gunpo, Korea) was used to immobilize samples on a membrane, and a cutting device (TBC-50Ts; Taewoo Co., Namyangju, Korea) was used to cut the membranes. A field emission scanning electron microscope (Hitachi S-4700, Tokyo, Japan) and a light microscope (Olympus BX43, Tokyo, Japan) were used to characterize the structure of PVA tape. An XPR ultra-microbalance (XPR6UD5©; Mettler Toledo, Masstron, OH, USA) was used to weigh tapes. All signals from the strips were measured and analyzed with a ChemiDoc XPS+ imaging system and Image lab software (6.0.1; Bio-Rad Laboratories), respectively. The ELISA results were evaluated using a cell imaging multi-mode reader (Cytation 5).