SERS-Temperature Dual-Mode T-type Lateral Flow Strip for Accurate Detection of Free and Total Prostate-Specific Antigens in Blood.

Accurate point-of-care (POC) analysis of cancer markers is the essence in the comprehensive early screening and treatment of cancer. Dual-mode synchronous detection is one of the effective approaches to reduce the probability of false negatives or false positives. As a result, this can greatly improve the accuracy of diagnosis. In this work, a surface-enhanced Raman scattering (SERS)-temperature dual-mode T-type lateral flow strip was fabricated to direct and simultaneous POC detection of total and free prostate-specific antigens (t-PSA and f-PSA) in blood. With the advantage of high stability of T-type lateral flow strip and simultaneous acquirement of assay results for t-PSA and f:t PSA ratio, the proposed method has high accuracy in the diagnosis of prostate cancer, especially in the diagnostic gray zone between 4.0 and 10.0 ng/mL. The SERS-temperature dual-signal has a good linear correlation with either f-PSA or t-PSA. To evaluate the clinical diagnostic performance of the proposed method, spiked human serum samples and the whole blood sample were analyzed. The assay results showed good recovery, and compared with traditional electrochemiluminescence immunoassay (ECLIA) method (t-PSA: 43.151; f/t ratio: 0.08), the results obtained by the proposed method were similar (t-PSA: 40.15 (SERS), 36.21 (temperature); f/t ratio: 0.08 (SERS), 0.08 (temperature), but the detection time (15 min) and cost ($0.05) had been greatly reduced. Therefore, the proposed SERS-temperature synchronous dual-mode T-type lateral flow strip has a strong application potential in the field of accurate large-scale diagnostics of prostate cancer on-site by simultaneous POC detection of t-PSA and f-PSA in blood.

On-site preparation of sandwich plasmonic coupled SERS tape toward pesticide residue determination on food surface.

A sandwich plasmonic coupled surface enhanced Raman spectroscopy (SERS) tape is proposed prepared by peeling the chemical printed silver nanocorals (AgNCs) from Cu sheet with adhesive tape, which can sample targets from food surface and sandwich them between substrates and Cu sheet for SERS detection. The solid-to-solid transformation method for fabricating SERS tapes can effectively avoid the weakening of tape stickiness during the preparation process. The sandwich plasmonic coupled structure of AgNC substrate, targets, and Cu sheet display excellent SERS activity (EF = 1.62 × 107) for sensitive determination of analytes. In addition, due to the high heat conductivity of Cu sheet, the thermal effect of laser irradiation during SERS detection cannot damage the AgNC tapes, which ensures the reproducibility of subsequent quantification. The sandwich plasmonic coupled SERS tape is demonstrated to quantify malachite green (MG) and methyl parathion (MP) with good linear coefficients (> 0.98) by two typical calibration plots under different concentration ranges. The limit of detection (LOD) of the method is 0.17 ng/cm2 and 0.48 µg/cm2 (S/N = 3) for MG and MP. This method can realize the quantitative determination of MP and MG on the surface of fruits and fish scale with recoveries of 93-113%. The satisfactory detection results demonstrate the proposed sandwich plasmonic coupled AgNC tape can be successfully applied to SERS-based point-of-care testing (POCT) for pesticide residue determination, which will provide a new path for designing and constructing SERS tapes.

One-Step, On-Site Chemical Printing of a 3D Plasmon-Coupled Silver Nanocoral Substrate toward SERS-Based POCT.

Surface-enhanced Raman scattering (SERS) with the advantages of high sensitivity, nondestructive analysis, and a unique fingerprint effect shows great potential in point-of-care testing (POCT). However, SERS faces challenges in rapidly constructing a substrate with high repeatability, homogeneity, and sensitivity, which are the key factors that restrict its practical applications. In this study, we propose a one-step chemical printing strategy for synthesizing a three-dimensional (3D) plasmon-coupled silver nanocoral (AgNC) substrate (only need about 5 min) without any pretreatments and complex instruments. The galvanic replacement between AgNO3 and Cu sheets will provide both Ag0 for the formation of silver nanostructures and Cu2+ for the polymerization of fish sperm DNA (FSDNA). The protection of AgNCs is facilitated by the crosslinked FSDNA, which can improve the stability of the substrate and promote the control of its coral-like morphology. The obtained substrate displays excellent capacity of signal enhancement due to the 3D plasmon coupling both between nanocoral tentacles and between nanocorals and Cu sheets as well. Therefore, the AgNC substrates display high activity (enhancement factor = 1.96 × 108) and uniformity (RSD < 6%). Food colorants have been widely used in various foods to improve their color, but the inevitable toxicity of colorants seriously threatens food safety. Therefore, the proposed AgNC substrates were used to directly quantify three kinds of weak-affinity food colorant molecules including Brilliant Blue, Allura Red, and Sunset Yellow assisted by the capture by cysteamine hydrochloride (CA), showing the detection limits (S/N = 3) of 0.053, 0.087, and 0.089 ppm, respectively. The SERS method has been further applied in the detection of the three kinds of food colorants in both complex food samples and urine with recoveries of 91-119%. The satisfactory detection results suggest that the facile preparation strategy of AgNC substrates will be widely used in SERS-based POCT to promote the development of food safety and on-site healthcare.

DNA-Condensed Plasmonic Supraballs Transparent to Molecules.

DNA nanotechnology offers an unrivaled programmability of plasmonic nanoassemblies based on encodable Watson-Crick basepairing. However, it is very challenging to build rigidified three-dimensional supracolloidal assemblies with strong electromagnetic coupling and a self-confined exterior shape. We herein report an alternative strategy based on a DNA condensation reaction to make such structures. Using DNA-grafted gold nanoparticles as building blocks and metal ions with suitable phosphate affinities as abiological DNA-bonding agents, a seedless growth of spheroidal supraparticles is realized via metal-ion-induced DNA condensation. Some governing rules are disclosed in this process, including kinetic and thermodynamic effects stemming from electrostatic and coordinative forces with different interaction ranges. The supraballs are tailorable by adjusting the volumetric ratio between DNA grafts and gold cores and by overgrowing extra gold layers toward tunable plasmon coupling. Various appealing and highly desirable properties are achieved for the resulting metaballs, including (i) chemical reversibility and fixation ability, (ii) stability against denaturant, salt, and molecular adsorbates, (iii) enriched and continuously tunable plasmonic hotspots, (iv) permeability to small guest molecules and antifoulingness against protein contaminates, and (v) Raman-enhancing and photocatalytic activities. Innovative applications are thus foreseeable for this emerging class of meta-assemblies in contrast to what is achieved by DNA-basepaired ones.

Gold nanoparticle dimer-based immunochromatography for in situ ultrasensitive detection of porcine epidemic diarrhea virus.

The low detection sensitivity of lateral-flow immunochromatography assay (LFIA) based on spherical gold nanoparticle (AuNP) limits its wide applications. In the present study, AuNP dimers with strong plasma scattering and robust signal output were synthesized via the Ag ion soldering (AIS) strategy and used as labeled probes in LFIA to boost the sensitivity without any extra operation process and equipment. The established LFIA exhibited high sensitivity with a limit of detection (LOD) of 2.0 × 102 TCID50/mL for PEDV, which provides 50 times higher sensitivity than commercial LFIA based on spherical colloidal gold. In addition, the AuNP dimer-based LFIA showed strong specificity, good reproducibility, high stability, and good accordance to reverse transcription polymer chain reaction (RT-PCR) when detecting 109 clinical samples. Thus, the AuNP dimers is a promising probe for LFIA and the developed AuNP dimer-based LFIA is suitable for the rapid detection of PEDV in the field.

Steering DNA Condensation on Engineered Nanointerfaces.

DNA has received increasing attention in nanotechnology due to its ability to fold into prescribed structures. Different from the commonly adopted base-pairing strategy, an emerging class of amorphous DNA materials are formed by DNA's abiological interactions. Despite the great successes, a lack of nanoscale nucleation/growth control disables more advanced considerations. This work aims at harnessing the heterogeneous nucleation of metal-ion-glued DNA condensates on nanointerfaces. Upon unveiling key orthogonal factors including solution pH, ionic cross-linkers, and surface functionalities, chemically programmable DNA condensation on nanoparticle seeds is achieved, resembling a famous Stöber process for silica coating. The nucleation rules discovered on individual nanoseeds can be passed on to their dimeric assemblies, where broken spherical symmetry and the existence of interparticle gaps help a regiospecific DNA gelation. The steerable DNA condensation, and the multifunctions from DNA, metal ions, and nanocores, hold a great promise in noncanonical DNA nanotechnology toward novel applications.

Surface-Enhanced Electrochemiluminescence Imaging for Multiplexed Immunoassays of Cancer Markers in Exhaled Breath Condensates.

Recently we have demonstrated that the surface plasmon of noble metal nanoparticles can effectively enhance the ECL intensity of Ru(bpy)32+, and we named this detection principle as surface-enhanced electrochemiluminescence (SEECL-I). However, SEECL based on photomultiplier tube (PMT) detection can only detect one target at a time, which is not suitable for multiple targets detection. In this work, we combined our previous developed SEECL with a bioimaging device to develop a novel multiplexed immunassay for simultaneous and fast analysis of cancer markers. A core-shell nanocomposite consisted of gold-silicon dioxide nanoparticles doped with Ru(bpy)32+(Au@SiO2-Ru) with strong ECL emission was employed as ECL label due to the localized surface plasmon resonance (LSPR) of AuNPs, which can significantly enhance the ECL emission of Ru(bpy)32+. The ECL signals from the 4 × 4 electrode arrays were collected using the constant potential method (current-time curve method) imaging with a sCOMS camera. As a proof-of-concept application, we demonstrated the use of the proposed SEECL-I for simultaneous detection of carcinoembryonic antigen (CEA), neuron specific enolase (NSE), and squamous cell carcinoma antigen (SCC) in exhaled breath condensates (EBCs) with low detection limit (LOD) of 0.17, 0.33, and 0.33 pg/mL (S/N = 3), respectively. The results demonstrated that the proposed SEECL-I strategy can provide a high sensitivity, fast analysis, and high-throughput platform for clinical diagnosis of cancer markers in EBCs.

Research on Dynamic Measurement Method of Flow Rate in Tea Processing.

Tea flow rate is a key indicator in tea production and processing. Due to the small real-time flow of tea leaves on the production line, the noise caused by the transmission system is greater than or close to the real signal of tea leaves. This issue may affect the dynamic measurement accuracy of tea flow. Therefore, a variational mode decomposition combined with a wavelet threshold (VMD-WT) denoising method is proposed to improve the accuracy of tea flow measurement. The denoising method of the tea flow signal based on VMD-WT is established, and the results are compared with WT, VMD, empirical mode decomposition (EMD), and empirical mode decomposition combined with wavelet threshold (EMD-WT). In addition, the dynamic measurement of different tea flow in tea processing is carried out. The result shows that the main noise of tea flow measurement comes from mechanical vibration. The VMD-WT method can effectively remove the noise in the tea dynamic weighing signal, and the denoising performance is better than WT, VMD, EMD, and EMD-WT methods. The average cumulative measurement accuracy of the tea flow signal based on the VMD-WT algorithm is 0.88%, which is 55% higher than that before denoising. This study provides an effective method for dynamic and accurate measurement of tea flow and offers technical support for digital control of the tea processing.

Oil-Free Gold Nanobipyramid@Ag Microgels as a Functional SERS Substrate for Direct Detection of Small Molecules in a Complex Sample Matrix.

Surface-enhanced Raman scattering (SERS) is a super-sensitive analysis technology based on the target molecular fingerprint information. The enhancement of local electromagnetic field of the SERS substrate would increase the target molecules' Raman intensity which adsorb on the surface of nanoparticles. However, the existing adhesive macromolecules in the complex mixed sample would interfere with the adsorption of small target molecules, and it weakens the Raman intensity of target molecules. Microgels are one of the potential materials to suppress the interference of adhesive macromolecules and to avoid the complex pretreatments. However, most of the current microgel synthesis methods involve complex operations with precise instrumentation or the interference of oil and organic reagents. In this work, a simple and oil-free method was proposed to synthesize the gold nanobipyramid (Au NBP)@Ag@hyaluronic acid microgel via the condensation reaction of carboxyl and amino groups. As a proof-of-concept demonstration for small-molecule detection, the rhodamine 6G (R6G) molecules were allowed to enter inside the microgel through the meshes and adsorb on the surface of Au NBP@Ag nanoparticles within 30 min, while the macromolecule (bovine serum albumin in this case) was retained outside the microgel in the meantime. In addition, under the combined action of lightning rod effect of Au NBP and surface plasmon resonance effect of silver render the microgels with high SERS activity. The synthetic Au NBP@Ag@hyaluronic acid microgels were applied to detect 6-thioguanine in the human serum without any pretreatment process, and it showed a high signal enhancement and stable SERS signal, which can satisfy the requirement of clinical diagnosis. These results show that the proposed microgels have potential applications in the field of point-of-care testing.

A universal strategy for the incorporation of internal standards into SERS substrates to improve the reproducibility of Raman signals.

The uneven distribution of metal nanoparticles is a vital influencing factor in the poor uniformity of surface-enhanced Raman scattering (SERS) substrates, which is a challenge in SERS quantitative analysis. Recent reports showed that the reproducibility of a nonuniform SERS substrate can be effectively improved by the use of an internal standard (IS). However, most of these approaches require the investment of time for precise regulation, and those approaches based on the addition of an IS are specific to a certain substrate. In this work, we proposed a simple, rapid and universal method to incorporate an IS into a SERS substrate for improving the reproducibility of Raman signals based on the systematic evaluation of the influencing factors of the competitive adsorption between the IS and the target analytes. Following the proposed pressure drop-coating (PDC) method, an IS-modified gold nanobipyramids (Au NBPs)/anodic aluminum oxide (AAO) SERS substrate was fabricated within 1 min, showing high reproducibility of Raman signals. In addition, the IS-modified Au NBPs/AAO SERS substrate was successfully applied to analyze thiram in freshly squeezed apple juice and the result showed a stable Raman signal with a relative standard deviation of less than 6.00%. What is more, three different commercial SERS chips were modified with an IS molecule using the PDC method. Compared to the traditional SERS chips, the Raman signal reproducibility of the functionalized SERS chips was improved significantly. Since the addition of an IS is not based on a certain substrate, the proposed approach could be useful for all the researchers working in the field of SERS.

DW14383 is an irreversible pan-FGFR inhibitor that suppresses FGFR-dependent tumor growth in vitro and in vivo.

Fibroblast growth factor receptor (FGFR) is a promising anticancer target. Currently, most FGFR inhibitors lack sufficient selectivity and have nonnegligible activity against kinase insert domain receptor (KDR), limiting their feasibility due to the serious side effects. Notably, compensatory activation occurs among FGFR1-4, suggesting the urgent need to develop selective pan-FGFR1-4 inhibitors. Here, we explored the antitumor activity of DW14383, a novel irreversible FGFR1-4 inhibitor. DW14383 exhibited equivalently high potent inhibition against FGFR1, 2, 3 and 4, with IC50 values of less than 0.3, 1.1, less than 0.3, and 0.5 nmol/L, respectively. It is a selective FGFR inhibitor, exhibiting more than 1100-fold selectivity for FGFR1 over recombinant KDR, making it one of the most selective FGFR inhibitors over KDR described to date. Furthermore, DW14383 significantly inhibited cellular FGFR1-4 signaling, inducing G1/S cell cycle arrest, which in turn antagonized FGFR-dependent tumor cell proliferation. In contrast, DW14383 had no obvious antiproliferative effect against cancer cell lines without FGFR aberration, further confirming its selectivity against FGFR. In representative FGFR-dependent xenograft models, DW14383 oral administration substantially suppressed tumor growth by simultaneously inhibiting tumor proliferation and angiogenesis via inhibiting FGFR signaling. In summary, DW14383 is a promising selective irreversible pan-FGFR inhibitor with pan-tumor spectrum potential in FGFR1-4 aberrant cancers, which has the potential to overcome compensatory activation among FGFR1-4.

Discovery of a potent tyrosine kinase AXL inhibitor bearing the 3-((2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yl)amino)pyrazine core.

Starting from the recently launched FLT3/AXL multi-targeted inhibitor Gilteritinib (5), we conducted a side-chain ring closure medicinal chemistry approach leading to the identification of compound 15c as a highly potent AXL inhibitor in the biochemical and cellular anti-proliferative assays, with IC50 values of 1.2 and 0.3 nM, respectively. Compared with the reference compound 5, our new discovered AXL inhibitor 15c is more potent in both assays.

Nanosecond-Laser-Based Charge Transfer Plasmon Engineering of Solution-Assembled Nanodimers.

The ability to re-engineer self-assembled functional structures with nanometer accuracy through solution-processing techniques represents a big challenge in nanotechnology. Herein we demonstrate that Ag+-soldered nanodimers with a steric confinement coating of silica can be harnessed to realize an in-solution nanosecond laser reshaping to form interparticle conductive pathway with finely controlled conductance. The high structural purity of the nanodimers, the rigid silica coating, and the uniform (but still adjustable) sub-1-nm interparticle gap together determine the success of the laser reshaping process. This method is applicable to DNA-assembled nanodimers, and thus promises DNA-based programming toward higher structural complexity. The resulting structures exhibit highly tunable charge transfer plasmons at visible and near-infrared frequencies dictated by the fluence of the laser pulses. Our work provides an in-solution, rapid, and nonperturbative route to realize charge transfer plasmonic coupling along prescribed paths defined by self-assembly, conferring great opportunities for functional metamaterials in the context of chemical, biological, and nanophotonic applications. The ability to continuously control a subnm interparticle gap and the nanomeric width of a conductive junction also provides a platform to investigate modern plasmonic theories involving quantum and nonlocal effects.

Freeze the Moment: High Speed Capturing of Weakly Bonded Dynamic Nanoparticle Assemblies in Solution by Ag Ion Soldering.

Despite the versatile forms of colloidal aggregates, these spontaneously formed structures are often hard to find a suitable application in nanotechnology and materials science. A determinate reason is the lack of a suitable method to capture the transiently formed and quickly evolving colloidal structures in solution. To address this challenge, a simple but highly efficient strategy is herein reported to capture the dynamic and metastable colloidal assemblies formed in an aqueous or nonaqueous solution. This process takes advantage of a recently developed Ag ion soldering reaction to realize a rapid fixation of as-formed metastable assemblies. This method works efficiently for both solid (3D) nanoparticle aggregates and weakly bonded fractal nanoparticle chains (1D). In both cases, very high capturing speed and close to 100% efficiency are achieved to fully retain a quickly growing structure. The soldered nanochains further enable a fabrication of discrete, uniform, and functionalizable nanoparticle clusters with enriched linear conformation by mechanical shearing, which would otherwise be difficult to make. The captured products are water dispersible and mechanically robust, favoring an exploration of their properties toward possible applications. The work paves a way to previously untouched aspects of colloidal science and thus would create new chances in nanotechnology.

[Intervention effect of Modified Dahuang Zhechong Granule on epididymal morphological changes in experimental varicocele rats].

OBJECTIVE: To explore the effect of Modified Dahuang Zhechong Granule (MDZG) on the development and maturation of epididymal sperm in experimental varicocele (VC) rats. METHODS: Sixty SD male rats were randomly divided into six groups of equal number, sham operation, VC model, Aescuven forte, and low-, medium- and high-dose MDZG. The model of left VC was made by the Turner method in all the rats except those of the sham operation group, followed by treatment with 0.9% normal saline for the animals in the sham operation and VC model groups, Aescuven forte tablets at 54 mg per kg of the body weight for those in the Aescuven forte group, and MDZG at 0.6, 1.2 and 2.4 g/ml for those in the low-, medium- and high-dose MDZG groups, all administered intragastrically qd for 8 successive weeks. Then, all the rats were sacrificed and their left epididymides harvested for examination of the quality of the epididymal sperm and the local microscopic and ultrastructural changes of the epididymal tissue. RESULTS: The VC model rats showed significant apoptosis of the epididymal sperm cells, interstitial edema, microvascular dilatation, degeneration and degeneration of the epithelial cells, degeneration of some principal cells and basal cell vacuoles, and immature spermatids in the lumen. Sperm motility was significantly increased in the Aescuven forte and low-, medium- and high-dose MDZG groups as compared with the VC models (P <0.01). Both sperm concentration and motility were markedly higher in the high-dose MDZG than in the Aescuven forte group (P <0.05). Remarkable apoptosis of epididymal sperm cells was observed in the microenvironment of sperm development in the VC models, which exhibited no statistically significant difference from that in the rats of the medium- and high-dose MDZG groups. CONCLUSIONS: Experimental varicocele induced local apoptosis of epididymal sperm cells, interstitial edema and microvascular dilatation in the rat epididymis, while Modified Dahuang Zhechong Granule could improve the stability of epididymal sperm maturation and contribute to their development.

Laser damage dependence on the size and concentration of precursor defects in KDP crystals: view through differently sized filter pores.

We investigate the laser-induced damage performance at 1064 nm of potassium dihydrogen phosphate (KDP) crystals grown using filters of different pore sizes. The aim is to explore a novel method for understanding laser-matter interactions with regard to physical parameters affecting the ability of damage precursors to initiate damage. By reducing the pore size of filters in continuous filtration growth, we can improve laser damage resistance. Furthermore, we develop a model based on a Gaussian distribution of precursor thresholds and heat transfer to obtain a size distribution of the precursor defects. Smaller size and/or lower concentration of precursor defects could lead to better damage resistance.

[Evaluation for extraction process of Shenqi Tongmai Yizhi particles based on antioxidant capacity in vitro and its spectrum-effect relation].

Based on DPPH method, the antioxidant activities of Shenqi Tongmai Yizhi particles with different extraction processes were compared. The contribution to the anti-oxidant capacity in vitro was explored by means of grey relational analysis on different chemical compositions in the fingerprint. The results showed that the IC50 concentration values of water extract, water extract from alcohol precipitation, alcohol extract, and alcohol and water extract were 0.801 4, 0.859 1, 0.796 1, 0.918 0 g•L⁻¹; and the alcohol extract is the best method to extract antioxidative components, with the highest antioxidant activity and lowest IC50. When the mass concentration of the herbs reached a certain degree, its free radical clearance rate was similar to that of vitamin C control group. The order of different chemical contributions of constituents to the antioxidant activity in the fingerprint was 4>3>33>53>9>10>11>34>15>59>8>61>52>20>42>18>29. The preliminary exploration for the spectrum efficiency relations provides reference for studying traditional Chinese medicine compound processing method and the pharmacodyamic material basis.

Dry Sintering Meets Wet Silver-Ion "Soldering": Charge-Transfer Plasmon Engineering of Solution-Assembled Gold Nanodimers From Visible to Near-Infrared†I and II†Regions.

Achieving highly tunable and localized surface plasmon resonance up to near infrared (NIR) regions is a key target in nanoplasmonics. In particular, a self-assembly process capable of producing highly uniform and solution-processable nanomaterials with tailor-made plasmonic properties is lacking. We herein address this problem through a conjunctive use of wet Ag+ soldering and dry thermal sintering to produce nanodimer-derived structures with precisely engineered charge-transfer plasmon (CTP). The sintered dimers are water soluble, featuring gradually shifted CTP spanning an 800 nm wavelength range (up to NIR II). Upon silica removal, the products are grafted by DNA to offer surface functionality. This process is also adaptable to DNA-linked AuNP dimers toward plasmonic meta-materials via DNA-guided soldering and sintering.

Modeling the effect of nanosecond laser conditioning on the femtosecond laser-induced damage of optical films.

The effect of nanosecond laser conditioning on the femtosecond laser-induced damage behaviors of Al2O3, HfO2, SiO2 single layers and Al2O3/SiO2 high reflectors (HR) are explored. During femtosecond laser damage test, negative effects on enhancing the femtosecond laser-induced damage threshold (LIDT) of optical films after the nanosecond laser conditioning is found, which is opposite to the LIDT improvement in the nanosecond range. To explain the mechanism after nanosecond laser conditioning, a theoretical model including multiphoton ionization (MPI), avalanche ionization (AI) and decays of electrons with one defect state is built to simulate the evolution of electron density in the conduction band. A permanent mid-gap defect state resulting from the process of laser conditioning is introduced in our model, which is found to contribute seed electrons to conduction band and hence accelerate the final breakdown. Both the experimental result and theoretical calculation agree very well with each other.

Mitigation of scattering defect and absorption of DKDP crystals by laser conditioning.

The variation of scattering and absorption in DKDP crystals by laser conditioning was investigated by combining light scattering technique and on-site transmittance measurement technique. Laser-induced disappearance of scattering defects was observed, and variation of transmittance was achieved. Using Mie theory, a kind of absorbing defects, aside from scattering defect, was discovered. Moreover, the experimental results demonstrated that the absorption of crystal could be mitigated by laser conditioning.