High Specific Recognition Research Articles

LAMP assay coupled CRISPR/LbCas12a system in a single-tube method for visual detection of meat adulteration

Meat adulteration frequently infringes upon consumers' rights and interests, disrupts the market, and violates religious beliefs. However, implementing on-site detection of meat samples remains technically challenging for widespread applications. Here, we developed a point-of-care testing (POCT) platform for fast, highly sensitive, and on-site detection of meat adulteration, based on loop-mediated isothermal amplification (LAMP) coupled the clustered regularly interspaced short palindromic repeats (CRISPR) system in a single-tube. This platform integrates the efficient exponential amplification capacity of LAMP and the high specificity recognition capability of CRISPR, reducing the false positive results and minimizing LAMP aerosol contamination risk. Pork was selected as the target for adulteration detection, qualitative detection by the naked eye can be achieved within 1 h from sample collection to results with a sensitivity as high as 0.05 % to detect binary mixtures of pork in beef and the limit of DNA detection was at 50 pg/μL which 10 times higher than the individual LAMP assay. This work is expected to offer a powerful universal POCT platform for addressing a range of meat adulteration issues, suitable for enhancing food safety measures.

Integrated microfluidic chip with a MIP-gated organic electrochemical transistor for continuous, selective, and label-free quantification of tumor marker

Early diagnosis of cancer is crucial to effective treatment of the patients and to higher survival rates. Tumor marker detection as an effective and noninvasive cancer diagnosis method has important clinical significance. N1, N12-diacetylspermine (DAS) is a new tumor marker. The development of its detection methods is an urgent need for medical diagnosis and human health monitoring. The conventional detection methods of DAS are labor-intensive and time-consuming approaches. In this work, a novel electrochemical detection platform was established by integrating an organic electrochemical transistor (OECT) with a microfluidic chip and applied for trace measurement of DAS. The sensing unit is a gate electrode in the OECT modified with molecularly imprinted polymers (MIPs), which can be used as an artificial receptor to realize label-free detection. The high specific recognition ability of MIPs films combined with the amplified function of an OECT yielded a highly sensitive and selective electrochemical detection chip. The integrated microfluidic channel controlledthe exact amount of solution to be delivered and provided real-time detection of DAS. The experimental results indicated that changes in channel current was proportional to DAS concentrations from 1 nM to 10 μM, with the detection limits of 0.8 nM. It is the first demonstration of the integration of a microfluidic system with a MIP-gated OECT for real-time tumor marker detection. The study results of this work helped to develop Point-Of-Care (POC) tools for the early diagnosis of lung cancer, colon cancer and breast cancer.

Self-Healing B ← N-Based Hydrogen-Bonded Organic Framework for Exclusive Recognition and Separation of Toluene from Methyl-Cyclohexane.

The effective separation of aromatic and aliphatic hydrocarbons remains a notable challenge in the petrochemical industry. Herein, we report a self-healing three-dimensional B ← N-based hydrogen-bonded organic framework (HOF), BN-HOF-1, constructed from discrete B ← N inclusive dimers through weak C-H···F and C-H···N hydrogen-bonding interactions. To make use of the specific recognition of the B ← N inclusive dimers for the toluene molecules and the reversible ad/desorption nature of this novel HOF, BN-HOF-1 can exclusively recognize and separate toluene from the mixtures of toluene-methylcyclohexane, thus generating 99.6% pure toluene from its mixtures after gentle heating, the recorded value among any reported materials for toluene purification. After the toluene molecules were released from the framework, it becomes the condensed BN-HOF-1a, which can be further reused for the highly selective recognition and purification of toluene from its binary mixtures, through the reversible structural recovery back to BN-HOF-1. Single-crystal X-ray diffraction and molecular modeling studies reveal that the high specific toluene recognition is attributed to the complementary electrostatic potential between the host B ← N inclusive dimers and the guest toluene, while the self-healing and recovery nature of this HOF is attributed to weak intermolecular hydrogen-bonding interactions.

Rational design of molecularly imprinted electrochemical sensor based on Nb2C-MWCNTs heterostructures for highly sensitive and selective detection of Ochratoxin a

Developing an efficient method for screening Ochratoxin A (OTA) in agriculture products is vital to ensure food safety and human health. However, the complex food matrix seriously affects the sensitivity and accuracy. To address this issue, we designed a novel molecularly imprinted polymer (MIP) electrochemical sensor based on multiwalled carbon nanotube-modified niobium carbide (Nb2C-MWCNTs) with the aid of the density functional theory (DFT). In this design, a glassy carbon electrode (GCE) was first modified by Nb2C-MWCNTs heterostructure. Afterward, the MIP layer was prepared, with ortho-toluidine as a functional monomer selected via DFT and OTA acting as a template on the surface of Nb2C-MWCNTs/GCE using in-situ electropolymerization. Electrochemical tests and physical characterization revealed that Nb2C-MWCNTs improved the sensor's active surface area and electron transmission capacity. Nb2C-MWCNTs had a good synergistic effect on MIP, endowing the sensor with high sensitivity and specific recognition of OTA in complex food matrix systems. The MIP sensor showed a wide linear range from 0.04 to 10.0 μM with a limit of detection (LOD) of 3.6 nM. Moreover, it presented good repeatability and stability for its highly antifouling effect on OTA. In real sample analysis, the recoveries, ranging from 89.77% to 103.70%, agreed well with the results obtained by HPLC methods, suggesting the sensor has good accuracy and high potential in practical applications.

Electronic Effect Driven Specific and Sensitive Recognition toward GHB.

Precise detection of a trace substance that intrinsically possesses weak chemical activity and less-distinctive spatial structure is of great significance, but full of challenges, as it could not be effectively recognized via either an active covalent reaction process or multiple noncovalent interactions toward its simple structure. Here, the electronic-effect-driven recognition strategy was proposed to visually sense an illicit drug, γ-hydroxybutyric acid (GHB), which was treated as an analyte model due to its inherent simple structure. In particular, a sensing system composed of two probes substituted by the nitro (-NO2) and the hydrogen (-H), was constructed with the characteristic yellow coloring and blue fluorescence, as well as high sensitivity (0.586 ng/mL), fast response (0.2 s), and specific recognition, even in the presence of 22 interferents. In addition, a portable eyeshadow box-like sensing chip was fabricated and proven to be reliable and feasible in sensing GHB disguised in liquors for self-protection in a covert manner. Hence, this work developed an electronic-effect-driven modulation strategy of the recognition interaction between the probe and the analyte and, thus, would open up a new thought for detecting the analyte with weak activity and a simple structure, as well as propel the relevant application in real scenarios.

Construction of a New Probe Based on Copper Chaperone Protein for Detecting Cu2+ in Cells.

Biomacromolecular probes have been extensively employed in the detection of metal ions for their prominent biocompatibility, water solubility, high selectivity, and easy modification of fluorescent groups. In this study, a fluorescent probe FP was constructed. The probe FP exhibited high specificity recognition for Cu2+. With the combination of Cu2+, the probe was subjected to fluorescence quenching. The research suggested that the probe FP carried out the highly sensitive detection of Cu2+ with detection limits of 1.7 nM. The fluorescence quenching of fluorescamine was induced by Cu2+ perhaps due to the PET (photoinduced electron transfer) mechanism. The FP-Cu2+ complex shows weak fluorescence, which is likely due to the PET quenching effect from Cu2+ to fluorescamine fluorophore. Moreover, the probe FP can be employed for imaging Cu2+ in living cells. The new fluorescent probe developed in this study shows the advantages of good biocompatibility and low cytotoxicity. It can be adopted for the targeted detection of Cu2+ in cells, and it has promising applications in the mechanism research and diagnosis of Cu2+-associated diseases.

Ultrasensitive cancer cell sensing based on tapered optical fiber operating near the dispersion turning point

Cancer cell screening is very important for its treatment and prognosis. The screening techniques are required with high sensitivity and specific recognition. Here, an ultrasensitive tapered optical fiber (TOF) biosensor for cancer cell sensing is proposed. The unique property of the dispersion turning point (DTP) in the TOF is utilized to greatly improve the sensing sensitivity. Combined with numerical calculations, the TOF with the extremely high refractive index (RI) sensitivity (close to 18390 nm/RIU) near the DTP is achieved by precisely controlling the diameter of the TOF. In addition, biotin is used to functionalize the TOF for specific recognition of the biotin-overexpressing cancer cells. The sensitivity and selectivity of the TOF were demonstrated by the detection of the biotin receptor protein (streptavidin) and overexpressed cancer cells (lung epithelial adenocarcinoma cells, A549). The experiment results show that the TOF has a low limit of detection (LOD) for streptavidin and cancer cells, which are 28.73 pM and 49 cells/mL respectively. At the same time, the TOF showed the obvious response to another biotin-overexpressed cancer cell (breast cancer cells, 4T1), but is insensitive to normal cells (gastric mucosa cells, GES-1 and mouse epithelioid fibroblasts cells, L929). We believe that the proposed biosensor which exhibited extremely high sensitivity and good selectivity, will have potential application prospects in the field of chemical and biological detection.

Chlorine-doped MoS2 quantum dots embedded in a molecularly imprinted polymer for highly selective and sensitive optosensing of quercetin.

Chlorine-doped MoS2 quantum dots (Cl-MoS2 QDs) embedded in a SiO2 molecularly imprinted polymer (Cl-MoS2 QDs@SiO2@MIP) have been successfully synthesized and can be used for highly selective and sensitive optosensing of quercetin. The novel environmentally friendly sensor integrated the advantages of the Cl-MoS2 QDs and MIP, high sensitivity and specific recognition for quercetin. The as-fabricated sensor is used to detect trace amounts of quercetin, and its fluorescence intensity showed a good linear decline with the increasing concentration of quercetin from 2 ng mL-1 to 200 ng mL-1 with a detection limit of 1.2 ng mL-1 (S/N = 3). The Cl-MoS2 QDs@SiO2@MIP probe was employed to assay the content of quercetin of real onion extract with good performance, which is in fine agreement with the result obtained by high performance liquid chromatography. The developed Cl-MoS2 QDs@SiO2@MIP sensor exhibits promising potential in the detection of quercetin.

UiO66-based molecularly imprinted polymers with water-compatible deep eutectic solvent as functional monomer for purification of lysozyme from egg white.

Protein-templated molecularly imprinted polymers have limitations such as poor mass transfer, slow recognition kinetics, and difficulties in isolation and purification due to their large molecular sizes, complex structures, and flexible conformations. To address these limitations and obtain lysozyme (Lyz)-imprinted polymers, a molecularly imprinted polymer (UiO66@DES-MIPs) was prepared for the first timeby using Lyz as a template molecule, a metal-organic framework (UiO66-NH2) as a matrix, and a water-compatible deep eutectic solvent (DES) as a functional monomer. The introduction of UiO66-NH2 by the solvothermal method with a large specific surface area and favorable stability and resistance to environmental disturbances into the MIPs can reduce the "embedding" phenomenon and acquire a higher binding capacity and fast mass transfer. In addition, a water-soluble binary DES (1:2 molar ratio of choline chloride to 1,3 dimethylurea) prepared by a hydrothermal method as a functional monomer generates multiple forces with Lyz, increasing the hydrophilicity of UiO66@DES-MIPs and contributing to the formation and stabilization of the imprinted sites. Consequently, UiO66@DES-MIPs exhibited good selectivity, water compatibility, and fast adsorption equilibrium (the adsorption equilibrated at 243.87 ± 4.88mgg-1 in 90min). Besides, reusability experiments indicated that the UiO66@DES-MIPs could be recycled six times without obvious loss of adsorption capacity. The imprinting factor of UiO66@DES-MIPs is 3.67. The isolation and purification of Lyz from egg white confirmed the practicability of UiO66@DES-MIPs. The high adsorption capacity and specific recognition make this polymer a promising candidate for the isolation and purification of biological macromolecules.

Preparation of an imprinted monolith for field simultaneously selective separation and enrichment of nitrophenols and lead (II) ion in water samples

Imprinted materials have gained significant attention due to its highly specific recognition performance. However, there is no study that using molecule and ion as mixed templates to prepare hybrid imprinted material for simultaneously selective separation and enrichment of molecules and ion. Here, a new strategy employing organic molecule and heavy metal ion as mixed templates to prepare imprinted material was proposed. 2,4-Dinitrophenol (2,4-DNP) and Pb2+ were selected as model templates to in-situ synthesize adsorbent based on imprinted monolith (AIM) in pipette tip. The obtained AIMs were used as the extraction medium of home-made multiple channels in-tip microextraction device (MCMD) for field simultaneously selective entrapment of nitrophenols (NPs) and Pb2+. The selection of functional monomers was well verified with Density function theory. Under the optimal fabrication conditions, the AIM displayed high specific recognition capability towards 2,4-DNP and Pb2+ with imprinted factors 7.31 and 5.20, respectively, and corresponding adsorption capacities were as high as 27.8 mg/g and 20.3 mg/g, respectively. The practicality of proposed AIM/MCMD was evaluated by simultaneously on-site selective capture of NPs and Pb2+ in various environmental waters prior to quantification with HPLC (for NPs) and atomic absorption spectrometry (for Pb2+). Results well evidence that the proposed strategy is feasible in the preparation of imprinted polymer for simultaneously specific extraction of molecules and metal ion, and the developed AIM/MCMD is a useful field sample preparation method for reliable quantification of NPs and Pb2+ in aqueous samples.

Fluorescence/colorimetric dual-signal sensor based on rare-earth nanosheets for quantitative detection of dopamine

A novel fluorescence/colorimetric dual-output dopamine (DA) sensor based on rare-earth nanosheets was constructed. Layered europium hydroxide (NO3-LEuH) was as the precursor, and the organic light-emitting molecule 8-naphthylamine-1-naphthalene sulfonic acid (ANS) and the surface-active molecule 1-octane sulfonate sodium (OS) were as the guests to synthesize fluorescent nanocomposite OS0.2ANS0.8-LEuH with novel emission properties. The nanocomposite was dispersed in a mixed solvent of formamide and water to prepare single-layer nanosheets. Based on fluorescence and ultraviolet absorption analysis, it was confirmed that in the range of 0.001–0.2 mM, the DA concentration had a good positive linear relationship with the fluorescence quenching efficiency F0/F and absorbance, and the response detection limits of the fluorescence method and the colorimetric method were 0.29 μM and 0.058 μM, respectively. The sensor has high specificity recognition ability and can be used for the detection of actual samples, and the dual-signal output sensing strategy greatly improves the accuracy and reliability of detection.

SERS-active immunoassay kit for SARS-CoV‑2 mediated by the cooperative chemical and electromagnetic effects of MXene modified with gold nanowires

Surface-enhanced Raman scattering (SERS) technique with high sensitivity, reliable specificity, and rapid recognition ability exhibits attractive promise for the effective fast-monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, a novel SERS-active immunoassay kit for SARS-CoV-2 nucleocapsid (N) protein was prepared by in-situ growing gold (Au) nanowire forests (NFs) onto Ti3C2Tx, which was then modified onto polymethyl methacrylate (PMMA) matrix and encapsulated into kit. It was noted that the Au nanowires with fibrous structures which vertically anchored on Ti3C2Tx served as perfect channels to promote photo-induced charge transfer. The synergistic action of electromagnetic and chemical effects resulted in an enhancement factor (EF) of 1.27 × 107. Furthermore, the unreliable fluctuation of the enhanced signal was eliminated by using the intrinsic Raman signal of the flexible PMMA platform, achieving an improved correlation coefficient (R2) value from 0.950 to 0.990. Moreover, the as-designed immunoassay kit with both high sensitivity and remedied quantitative ability rendered by the Ti3C2Tx@Au NFs-PMMA composite exhibited a powerful performance in the practical detection of N-protein with concentration low to 5.0 × 10−8 mg/mL.

Electrospinning carbon fibers based molecularly imprinted polymer self-supporting electrochemical sensor for sensitive detection of glycoprotein

For glycoprotein detection that is both specific and sensitive in clinical diagnosis and treatment, it is necessary to develop artificial bionic recognition materials. By integrating boronate affinity controllable oriented surface imprinting with carbon nanofibers (CNF) as self-supported electrodes, a simple and cost-effective method for preparing glycoprotein-imprinted self-supported electrochemical biosensors was presented in this paper. The molecular imprinted polymers (MIPs) electrode based on Co, Mo2C-CNF was prepared in the presence of horseradish peroxidase (HRP) as a model glycoprotein by cyclic voltammetry. The CNF mats, composed of Co and Mo2C nanoparticles encapsulated in carbon nanofibers (Co, Mo2C-CNF) via electrospinning, could offer more electrical conductivity and active sites for the detection process. The results demonstrated that the hybrid structure of Co, Mo2C-CNF and MIPs exhibited high specificity and excellent selective recognition efficiency for HRP. The detection limit was obtained as 7.4 fg mL−1. The developed biosensor was applied to detect HRP in human serum with a recovery ranging from 89.21 % to 116.68 %. The developed MIPs electrochemical sensor has great potential for developing highly sensitive and specific glycoprotein imprinting biosensors.

Detection of TNT wastewater by the molecular imprinted porous membrane

In this paper, the preparation scheme of TNT molecularly imprinted porous film and the scheme of TNT identification and detection in wastewater are designed by means of molecular imprinting technology. The molecularly imprinted porous film material was prepared by the self-assembly method, and the TNT residue adsorption was detected by a UV-visible spectrophotometer. The concentration of TNT waste liquid was calculated by absorbance, and the effect of the deviation of ultraviolet spectra of different contents of TNT waste liquid was discussed. The results show that the molecularly imprinted films have high adsorption and specific recognition effect on TNT, which can be used for rapid and sensitive detection of TNT in the waste liquid.

Construction of hybridization chain reaction induced optical signal directed change of photonic crystals-DNA hydrogel sensor and its visual determination for aflatoxin B1.

Herein, we have introduced hybridization chain reaction (HCR) into the photonic crystals (PhCs) hydrogel, for the first time, realizing HCR for inducing the change of the optical signal of PhCs hydrogel and using this hydrogel as a sensor for determination of the aflatoxin B1 (AFB1). By using specific sequences as the cross-linker, the extension of the cross-linker by HCR drives the swelling of the hydrogel, and the optical property of 2D PhCs array converts this swelling into a change of the Debye diffraction ring. Moreover, by further selecting the aptamer to construct the cross-linker, the hydrogel is also endowed with a unique capability for AFB1, making the hydrogel a novel sensor based on the signal amplification strategy. The results show that the designed hairpin DNAs can effectively trigger the HCR and cause the swelling of hydrogel, and the hydrogel sensor has a good determination performance and high specific recognition for AFB1.

Molecularly imprinted polymers-isolated AuNP-enhanced CdTe QD fluorescence sensor for selective and sensitive oxytetracycline detection in real water samples

A molecularly imprinted polymers (MIPs)-isolated AuNP-enhanced fluorescence sensor, AuNP@MIPs-CdTe QDs, was developed for highly sensitive and selective detection of oxytetracycline (OTC) in aqueous medium. The developed sensor combined the advantages of strong fluorescence signal of metal-enhanced fluorescence (MEF), high selectivity of MIPs, and stability of CdTe QDs. The MIPs shell with specific recognition served as an isolation layer to adjust the distance between AuNP and CdTe QDs to optimize the MEF system. The sensor demonstrated the detection limit as low as 5.22 nM (2.40 μg/L) for a concentration range of 0.1–3.0 μM OTC and good recovery rates of 96.0–103.0% in real water samples. In addition, high specificity recognition for OTC over its analogs was achieved with an imprinting factor of 6.10. Molecular dynamics (MD) simulation was utilized to simulate the polymerization process of MIPs and revealed H-bond formation as the mainly binding sites of APTES and OTC, and finite-difference time-domain (FDTD) analysis was employed to obtain the distribution of electromagnetic field (EM) for AuNP@MIPs-CdTe QDs. The experimental results combined with theoretical analyses not only provided a novel MIP-isolated MEF sensor with excellent detection performance for OTC but also established a theoretical basis for the development of a new generation of sensors.

Highly sensitive and recognizable detection for trifluralin with alkyl-decorated fluorescent porous polymers

The sensing materials and portable devices with high sensitivity and specific recognition are urgently demanded for monitoring the pesticide residue in soil, water and food. Herein, two fluorescent porous polymers with carbazole-based chromophores decorated by n-butyls are synthesized through A3 + B2 and A3 + B4 condensation polymerizations, respectively. The hierarchical pores ranging from ultramicropore to mesopore are adjustable by varying the crosslinking density in the networks. The fluorescent quenching coefficient and limit of detection for trifluralin exceptionally reach 4.57 × 105 M−1 and 6.0 ppb, and the polymers exhibit ultra-sensitive recognizable ability for trifluralin in water. The quenching efficiencies for trifluralin in water, aqueous solutions of various cations and anions, vegetable juice and soil filtrate remain nearly the same. In particular, the fabricated polymer plates are portable and show visual fluorescent quenching for trifluralin solution at an extremely low concentration, and the plates can be easily recovered and repeatedly used. The results revealed in this manuscript are helpful for deeply understanding the effects of porosity parameters and alkyl-decorated chromophore groups on the sensitive and recognizable sensing mechanism for trifluralin.

Highly sensitive and facile microRNA detection based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and have been implicated in many diseases. Herein, we develop a target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system, which can achieve the ultrasensitive detection with simple operation and no annealing procedure. In this assay, T-ERCA combines the exponential amplification with rolling-circle amplification by introducing a dumb-bell probe with two enzyme recognition sites. miRNA-155 targets are activators that trigger exponential rolling circle amplification to produce large amounts of ssDNA, which is then recognized by CRISPR/Cas12a for further amplification. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Therefore, benefiting from the excellent amplification effect of T-ERCA and the high recognition specificity of CRISPR/Cas12a, the proposed strategy shows a wide detection range from 1 fM to 5 nM with a LOD (limit of detection) down to 0.31 fM. Moreover, it shows good application ability for assessing miRNA levels in different cells, indicating that the T-ERCA/Cas12a may provide a new guidance for molecular diagnosis and clinical practical application.

MiRNA-155 Biosensors Based on AlGaN/GaN Heterojunction Field Effect Transistors With an Au-SH-RNA Probe Gate

AlGaN/gallium nitride (GaN) heterojunction field effect transistors (HFETs) have been researched widely in the fields of gas detection and ions detection. In this article, we develop a biosensor based on AlGaN/GaN HFETs to detect the tumor biomarker miRNA-155 directly. By fixing the specific sulfhydryl modified ribonucleic acid (SH-RNA) probe on the Au-gate surface of the AlGaN/GaN HFET to fabricate an RNA-Au-AlGaN/GaN HFET biosensor, this biosensor can conveniently and rapidly detect miRNA-155. The experimental results show that this biosensor has an excellent detection limit of 1.81 fM and a high sensitivity of 77 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu $</tex-math> </inline-formula> A/log concentration with a linear range (2 fM–2 nM). The limit of detection is the lowest among the reported AlGaN/GaN HFET RNA biosensors. Meanwhile, this biosensor shows a high specific recognition capacity but is weak sensitive to the mismatched miRNA.

Promising Mass-Productive 4-Inch Commercial SERS Sensor with Particle in Micro-Nano Porous Ag/Si/Ag Structure Using in Auxiliary Diagnosis of Early Lung Cancer.

The construction of commercial surface enhanced Raman scattering (SERS) sensors suitable for clinical applications is a pending problem, which is heavily limited by the low production of high-performance SERS bases, because they usually require fine or complicated micro/nano structures. To solve this issue, herein, a promising mass-productive 4-inch ultrasensitive SERS substrate available for early lung cancer diagnosis is proposed, which is designed with a special architecture of particle in micro-nano porous structure. Benefitting from the effective cascaded electric field coupling inside the particle-in-cavity structure and efficient Knudsen diffusion of molecules within the nanohole, the substrate exhibits remarkable SERS performance for gaseous malignancy biomarker, with the limit of detection is 0.1 ppb and the average relative standard deviation value at different scales (from cm2 to µm2 ) is ≈16.5%. In practical application, this large-sized sensor can be further divided into small ones (1 × 1 cm2 ), and more than 65 chips will be obtained from just one 4-inch wafer, greatly increasing the output of commercial SERS sensor. Further, a medical breath bag composed of this small chip is designed and studied in detail here, which suggested high-specificity recognition for lung cancer biomarker in mixed mimetic exhalation tests.