Ultrasensitive Point‐of‐Care Test for Tumor Marker in Human Saliva Based on Luminescence‐Amplification Strategy of Lanthanide Nanoprobes

Abstract The point‐of‐care detection of tumor markers in saliva with high sensitivity and specificity remains a daunting challenge in biomedical research and clinical applications. Herein, a facile and ultrasensitive detection of tumor marker in saliva based on luminescence‐amplification strategy of lanthanide nanoprobes is proposed. Eu2O3 nanocrystals are employed as bioprobes, which can be easily dissolved in acidic enhancer solution and transform into a large number of highly luminescent Eu3+ micelles. Meanwhile, disposable syringe filter equipped with nitrocellulose membrane is used as bioassay platform, which facilitates the accomplishment of detection process within 10 min. The rational integration of dissolution enhanced luminescent bioassay strategy and miniaturized detection device enables the unique lab‐in‐syringe assay of tumor marker like carcinoembryonic antigen (CEA, an important tumor marker in clinic diagnosis and prognosis of cancer) with a detection limit down to 1.47 pg mL−1 (7.35 × 10−15 m). Upon illumination with a portable UV flashlight, the photoluminescence intensity change above 0.1 ng mL−1 (0.5 × 10−12 m) of CEA can be visually detected by naked eyes, which allows one to qualitatively evaluate the CEA level. Moreover, we confirm the reliability of using the amplified luminescence of Eu2O3 nanoprobes for direct quantitation of CEA in patient saliva samples, thus validates the practicality of the proposed strategy for both clinical diagnosis and home self‐monitoring of tumor marker in human saliva.

radiation ( = 0.154 nm). The Zeta potentials were measured with a Malvern Instrument (Nano ZS ZEN3600). Fourier-transform infrared (FTIR) spectra were recorded on a Magna 750 FTIR spectrometer. All fluorescence measurements were identified by a spectrometer equipped with both continuous (450 W) xenon and pulsed flash lamps (FLS980, Edinburgh Instruments).

Synthesis of Eu 2 O 3 nanocrystals (NCs):
Eu 2 O 3 NCs were synthesized from europium acetate precursors via a facile thermal decomposition route. [1] Briefly, 1 mmol of EuAc 3 ·4H 2 O were added into a 100 mL three-neck round-bottom flask containing 5 mL of OA, 5 mL of oleylamine, and 10 mL of 1-octadecene. The flask was then degassed with N 2 for 10 min at room temperature (RT).
After that, the reaction solution was heated to 150 °C for 1 h to dissolve the reactants and remove water. Subsequently, 1 mmol of Na 4 P 2 O 7 were added to the solution. After 10 min, the temperature was increased to 320 °C and maintained for 1 h, resulting in the formation of the Eu 2 O 3 NCs. The mixture was cooled down to RT. The Eu 2 O 3 NCs were precipitated by addition of ethanol, collected by centrifugation, washed with cyclohexane and ethanol several times, and finally re-dispersed in cyclohexane.

Synthesis of PAA-capped Eu 2 O 3 NCs:
We modified the surface of OA-capped NCs with polyacrylic acid (PAA) via ligand exchange. [2] 8.0 mL of DEG containing 0.5 g PAA was heated to 110 °C with vigorous stirring under N 2 flow. Then, 15 mL of cyclohexane solution containing 30 mg Eu 2 O 3 NCs was injected to the hot solution. The mixture was heated to 240 °C and kept at this temperature for 1 h until the solution became clear. After the solution was cooled down to RT, excess dilute hydrochloric aqueous solution was added. Finally, PAA-capped Eu 2 O 3 NCs were obtained by centrifugation, washed with pure water. The resulting powders can be well dispersed in water by ionizing the carboxylic groups with a dilute NaOH solution.

Calculation of photoluminescence (PL) quantum yield (QY):
The absolute PLQY was measured at RT by employing a barium sulfate coated integrating sphere (150 mm in diameter, Edinburgh) as the sample chamber that was mounted on the FLS980 spectrometer with the entry and output port of the sphere located in 90° geometry from each other in the plane of the spectrometer (excited by 365 nm light from xenon lamp). The PL emission intensity from 550 to 750 nm was integrated to calculate the absolute PLQY. A standard tungsten lamp was used to correct the optical response of the instrument. All the spectral data were corrected for the spectral response of both the spectrometer and the integrating sphere. We calculated the absolute PLQY based on the following equation:

S3
where N e and N a are the photons emitted and absorbed, respectively; L s is the emission intensity; E r and E s are the intensities of the excitation light in the presence of the pure hexane solution (reference) and the Eu 3+ micelles (sample) dispersed in hexane solution, respectively.

Synthesis of CEA PcAb-conjugated Eu 2 O 3 NCs:
The PAA-capped Eu 2 O 3 NCs can be easily connected to CEA PcAb by amidation reaction via NHS/EDC coupling. [3] In a typical process, 5 mg of sodium polyacrylate was dispersed in 1 mL aqueous solution containing 10 mg PAA-capped Eu 2 O 3 NCs. The mixture was stirred for 1 h at RT. After washing with distilled water for three times, 1 mL of PBS containing 1 mg NHS and 5 mg EDC was added for the activation of carboxyl with constant stirring for 30 min. Thereafter, 0.5 mg of CEA PcAb was added and incubated for 30 min. The resulting labeled NCs were purified by washing with PBS for three times and stored at 4 °C.

Preparation of the enhancer solution:
We prepared the enhancer solution with a modified formula as previously reported. [4] β-NTA  The assay of human saliva or serum samples was conducted following the same procedure by simply replacing the CEA standard solution with human saliva or serum samples. Every measurement was repeated three times and the CEA levels in human saliva or serum samples were determined by the calibration curve.

Qualitatively visual detection of CEA:
The incubation, labeling, washing and dissolution processes for CEA assay were conducted following the same procedure mentioned above. Upon excitation with a 365-nm UV flashlight, standard color card indicating CEA standard concentration was established with their corresponding fluorescence photographs. The assay of human saliva or serum samples was conducted following the same procedure by simply replacing the CEA standard solution with human saliva or serum samples. The CEA levels in human saliva or serum samples were evaluated according to the standard color card.
Statistical Analysis: All the assay experiments were repeated thrice independently unless otherwise indicated. Statistical analysis was compiled on the means of the data obtained from at least three independent experiments. The data of all assays were presented as the mean value ± standard deviation (SD). "n" numbers for each experiment are indicated in the figure legends. S5   Table S1. Comparison of the CEA levels in 20 human saliva samples independently determined by the luminescence-amplification strategy based on Eu 2 O 3 NCs and the commercial kit based on Eu 3+ -DTTA complex, respectively. The unit of the CEA levels is ng/mL. The CEA levels derived from Eu 2 O 3 nanoprobes are in good accordance with those measured by commercial kit, indicating that the proposed assay system is as reliable as that of commercial kit. Three independent experiments were carried out to yield the average value and deviation.

SUPPORTING INFORMATION
S7 Table S3. Assay precision and analytical recovery of CEA added to two saliva samples. The coefficient of variations (CVs) of all assays are lower than 7% and the recoveries are in the range of 92-106%. Both parameters well meet the acceptance criteria (CVs ≤ 15%; recoveries in the range of 90-110%) set for bioanalytical method validation. [6] Four independent experiments were carried out to yield the average value and deviation. These results demonstrate unambiguously the feasibility and reliability for monitoring the level of tumor markers in human saliva.  otherwise identical conditions. Statistical analysis was compiled on the means of the data obtained from three independent experiments. Upon excitation at 365 nm, the PL intensity of the the control experiment was found to be much lower than that of CEA assay.

SUPPORTING INFORMATION
S13 Figure S6. TR emission spectra of Eu 2 O 3 NCs in the enhancer solution for the assay of CEA in the synthetic saliva upon excitation at 365 nm. The red emission from Eu 3+ micelles was found to be in a gradient decrease along with the decreased CEA concentration.

Supporting Video Captions
Movie S1. Detailed procedures of CEA detection based on luminescence-amplification strategy of lanthanide nanoprobes.