Ultrasensitive fluorescent aptasensor for CRP detection based on the RNase H assisted DNA recycling signal amplification strategy

An aptamer-based method for the ultrasensitive fluorescence detection of C-reactive protein (CRP) was developed using the ribonuclease H (RNase H) assisted DNA recycling signal amplification strategy. In this assay, CRP can specifically bind to the aptamer of CRP and the DNA chain of P1 is released from the aptamer/P1 (Ap/P1) complexes. After the addition of the fluorescence labeled (5-FAM) RNA, P1 hybridizes with fluorescence labeled RNA to form a P1/RNA double strand. When RNase H is added, the RNA with fluorescence labeling in the double strand is specifically cut into nucleotide fragments, which cannot be adsorbed on the surface of the GO, so as to generate a fluorescence signal. In the absence of CRP, fluorescence labeled RNA cannot hybridize with P1 to form double strands, which is able to directly adsorb on the surface of GO, resulting in no fluorescence signal. The detection limit is as low as 0.01 ng mL⁻¹, with a linear dynamic range from 50 pg mL⁻¹ to 100 ng mL⁻¹. This sensor is able to detect CRP in spiked human serum, urine and saliva. Thus, it shows a great application prospect in disease diagnosis and prognosis.

An aptamer-based method for the ultrasensitive fluorescence detection of C-reactive protein (CRP) was developed using the ribonuclease H (RNase H) assisted DNA recycling signal amplification strategy.     

Ultrasensitive fluorescent aptasensor for MUC1 detection based on deoxyribonuclease I-aided target recycling signal amplification

A novel sensing strategy for sensitive detection of mucin 1 protein (MUC1) based on deoxyribonuclease I-aided target recycling signal amplification was proposed. In this paper, in the absence of MUC1, the MUC1 aptamer is absorbed on the surface of graphene oxide (GO) via π-stacking interactions. This results in quenching of the fluorescent label and no fluorescence signal is observed. Upon adding MUC1, the probe sequences could be specifically recognized by MUC1, leading to an increase in the fluorescence intensity. The detection limit is as low as 10 pg mL⁻¹, and a linear range from 50 pg mL⁻¹ to 100 ng mL⁻¹. The assay is specific and sensitive, and successfully applied to the determination of MUC1 in spiked human serum, urine and saliva. Importantly, the proposed aptasensing strategy has great potential in detecting various protein and even cancer cells.

A novel sensing strategy for sensitive detection of mucin 1 protein (MUC1) based on deoxyribonuclease I-aided target recycling signal amplification was proposed.     

Correction: A fluorescent biosensor for cardiac biomarker myoglobin detection based on carbon dots and deoxyribonuclease I-aided target recycling signal amplification

Correction for ‘A fluorescent biosensor for cardiac biomarker myoglobin detection based on carbon dots and deoxyribonuclease I-aided target recycling signal amplification’ by Jishun Chen et al., RSC Adv., 2019, 9, 4463–4468, https://doi.org/10.1039/C8RA09459D.

The authors are publishing this correction to draw the reader’s attention to three related papers that should have been cited as ref. 43–45 in this RSC Advances article. These have been given below as ref. 1–3, respectively.On page 4464, a citation to ref. 43 should be added to the end of the sentence beginning “Ultrapure water…”. Therefore, the sentence should be changed to “Ultrapure water obtained from a Millipore water purification system (18.2 MΩ cm resistivity, Milli-Q Direct 8) was used in all runs.⁴³”On page 4465, citations to ref. 43 and 44 should be added at the end of the sentence beginning “The fluorescence intensity…”. Therefore, the sentence should be changed to “The fluorescence intensity and the value of F₁/F₀ are selected to evaluate the effects of the reaction conditions on the sensing performance of the assay.^43,44”On page 4467, a citation to ref. 45 should be added to the end of the sentence beginning “Aptamer can recognize…”. Therefore, the sentence should be changed to “Aptamer can recognize MB specifically, and thus provide a promising method for its assay, this promising strategy might prove feasible as an MB assay for real samples.⁴⁵”The authors also regret that there are portions of unattributed text overlap in the Results and discussion and Conclusion sections with other papers, including ref. 43–45.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Carbon nanoparticle-protected RNA aptasensor for amplified fluorescent determination of theophylline in serum based on nuclease-aided signal amplification

A carbon nanoparticle (CNP) and Cryonase-aided method that realizes the amplified fluorescent detection of theophylline was proposed. The amplification technique exploits distinct binding affinities of CNP towards the FAM-labeled theophylline RNA aptamer (aptasensor) and aptasensor/theophylline complex as well as the protection effect of CNP for absorbed aptasensor from enzymatic digestion by Cryonase. Upon the addition of theophylline, it forms an aptasensor/theophylline complex with a fluorescent dye-tagged aptasensor that is initially absorbed and quenched by CNP. The nuclease activity of Cryonase towards detached aptasensor probes is then activated, leading to efficient cleavage of aptasensor probes and separation of the fluorescent dye from the CNP surface. Theophylline that has been liberated can launch another reaction cycle, which ensures the sensitivity enhancement. A detection limit is achieved as high as 6.3 nM, which is 400-fold better than traditional strategies. The proposed sensing system also provides desired selectivity even in serum samples. The assay is simple, sensitive, selective, and universal, and has great promise for the design and application of aptasensors in the biological, chemical, and biomedical fields.

A carbon nanoparticle (CNP) and Cryonase-aided method that realizes the amplified fluorescent detection of theophylline was proposed.     

A fluorescent biosensor for cardiac biomarker myoglobin detection based on carbon dots and deoxyribonuclease I-aided target recycling signal amplification

A sensitive biosensor using carbon dots and deoxyribonuclease I-aided target recycling signal amplification has been developed to detect myoglobin (MB), which is an important cardiac biomarker and plays a major role in the diagnosis of acute myocardial infarction (AMI). Here, in the absence of MB, the MB aptamer (Ap) is absorbed on the surface of carbon dots (CDs) through π–π stacking interactions, resulting in quenching of the fluorescent label by forming CD–aptamer complexes. Upon adding MB, the Ap sequences could be specifically recognized by MB, leading to the recovery of quenched fluorescence. Thus, quantitative evaluation of MB concentration has been achieved in a broad range from 50 pg mL⁻¹ to 100 ng mL⁻¹, and the detection limit is as low as 20 pg mL⁻¹. This strategy is capable of specific and sensitive detection of MB in human serum, urine, and saliva and can be used for the diagnosis of AMI in the future.

A sensitive biosensor has been developed to detect myoglobin (MB), which is an important cardiac biomarker and plays a major role in the diagnosis of acute myocardial infarction (AMI).     

Ultrasensitive DNA detection based on target-triggered rolling circle amplification and fluorescent poly(thymine)-templated copper nanoparticles

We describe a novel strategy for the ultrasensitive detection of target DNA based on rolling circle amplification (RCA) coupled with fluorescent poly(thymine)-templated copper nanoparticles (poly T-CuNPs). In the presence of target DNA, a padlock DNA probe that consists of two regions: a target DNA-specific region and a poly(adenine) region, is circularized by the ligation reaction, and the subsequent RCA reaction is promoted to generate long, concatemeric, single-stranded DNA (ssDNA) with a lot of repetitive poly T sequences. As a result, a large number of poly T-CuNPs are formed, exhibiting a highly fluorescent signal. However, in the absence of target DNA or in the presence of non-specific target DNA, the padlock DNA probe is not circularized and the subsequent RCA is not executed, leading to no production of fluorescent poly T-CuNPs. With this simple strategy, we successfully analyzed the target DNA with the ultralow detection limit of 7.79 aM, a value that is 3 or 7 orders of magnitude lower than those of previous RCA-based fluorescent DNA detection strategies. In addition, the developed system was demonstrated to selectively discriminate non-specific target DNAs with one-base mismatch, suggesting potential application in the accurate diagnosis of single nucleotide polymorphisms or mutations.

An ultrasensitive DNA detection method is developed based on target-triggered rolling circle amplification coupled with fluorescent poly(thymine)-templated copper nanoparticles.     

Ultrasensitive enzyme-free fluorescent detection of VEGF165 based on target-triggered hybridization chain reaction amplification

Sensitive detection of vascular endothelial growth factor (VEGF₁₆₅) is important for early cancer disease diagnosis in the clinic. A sensitive fluorescent sensing platform for VEGF₁₆₅ detection is developed in this work. It is based on a target-triggered hybridization chain reaction (HCR) and graphene oxide (GO) selective fluorescence quenching. In this assay, in the presence of the VEGF₁₆₅, the hairpin structure of Hp opens up and the initiation sequence will be exposed to Hp1 to open its hairpin structure. Then the opened Hp1 hybridizes with Hp2 to expose the complementary sequence of Hp1 which hybridizes with Hp1 again by HCR. Thus HCR would be initiated, generating super-long dsDNA. After the HCR, the double strands of the HCR product cannot be adsorbed on the GO surface. As a result, the HCR product gives a strong fluorescence signal which is dependent on the concentration of VEGF₁₆₅. By using VEGF₁₆₅ as a model analyte, the assay provides a highly sensitive fluorescence detection method for VEGF₁₆₅ with a detection limit down to 20 pg mL⁻¹. The proposed aptasensing strategy based on target-triggered HCR amplification can thus be realized. It was successfully applied to the determination of VEGF₁₆₅ in spiked human serum, urine and saliva. Therefore, it can easily have wide applications in the diagnosis of vital diseases.

Sensitive detection of vascular endothelial growth factor (VEGF₁₆₅) is important for early cancer disease diagnosis in the clinic.     

A signal-on fluorescent aptasensor based on gold nanoparticles for kanamycin detection

This study describes the development, verification and practical application of an aptasensor for the fluorometric detection of kanamycin. Using the nucleic acid aptamer with FAM fluorescent group as the conjugate, using gold nanoparticles as the fluorescence dynamic quenching source, a fluorescence sensor was fabricated through the signal-on method for the micro-detection of kanamycin. The nucleic acid chimera is connected to the fluorophore, and the gold nanoparticles are used as the fluorescence dynamic quenching source under actual conditions. The detection limit of kanamycin is 0.1 pM, and the detection range is 0.1 pM to 0.1 μM. This biosensor works satisfactorily in complex samples with no impurities, which gives this method an obvious advantage over other analytical methods. In addition, the mechanism of action between gold nanoparticles/FAM–aptamer/kanamycin is discussed and studied in depth here. It provides a more thorough analysis and more application possibilities for fluorescence-aptamer biosensing.

This study describes the development, verification and practical application of an aptasensor for the fluorometric detection of kanamycin.     

Enzyme-free ultrasensitive fluorescence detection of epithelial cell adhesion molecules based on a toehold-aided DNA recycling amplification strategy

Epithelial cell adhesion molecules (EpCAMs) play a significant role in tumorigenesis and tumor development. EpCAMs are considered to be tumor signaling molecules for cancer diagnosis, prognosis and therapy. Herein, an enzyme-free and highly sensitive fluorescent biosensor, with a combined aptamer-based EpCAM recognition and toehold-aided DNA recycling amplification strategy, was developed for sensitive and specific fluorescence detection of EpCAMs. Due to highly specific binding between EpCAMs and corresponding aptamers, strand a, which is released from the complex of aptamer/strand a in the presence of EpCAMs which is bound to the corresponding aptamer, triggered the toehold-mediated strand displacement process. An amplified fluorescent signal was achieved by recycling strand a for ultrasensitive EpCAM detection with a detection limit as low as 0.1 ng mL⁻¹, which was comparable or superior to that of reported immunoassays and biosensor strategies. In addition, high selectivity towards EpCAMs was exhibited when other proteins were selected as control proteins. Finally, this strategy was successfully used for the ultrasensitive fluorescence detection of EpCAMs in human serum samples with satisfactory results. Importantly, the present strategy may be also expanded for the detection of other targets using the corresponding aptamers.

A fluorescent biosensor with a combined aptamer-based EpCAM recognition and toehold-aided DNA recycling amplification strategy was developed.     

A one-step fluorescent biosensing strategy for highly sensitive detection of HIV-related DNA based on strand displacement amplification and DNAzymes

Sensitive and specific detection of HIV-related DNA is of great importance for early accurate diagnosis and therapy of HIV-infected patients. Here, we developed a one-step and rapid fluorescence strategy for HIV-related DNA detection based on strand displacement amplification and a Mg²⁺-dependent DNAzyme reaction. In the presence of target HIV DNA, it can hybridize with template DNA and activate strand displacement amplification to generate numerous DNAzyme sequences. With the introduction of Mg²⁺, DNAzyme can be activated to circularly cleave the substrate DNA, which leads to the separation of fluorophore reporters from the quenchers, resulting in the recovery of the fluorescence. Under the optimal experimental conditions, the established biosensing method can detect target DNA down to 61 fM with a linear range from 100 fM to 1 nM, and discriminate target DNA from mismatched DNA perfectly. In addition, the developed biosensing strategy was successfully applied to assay target DNA spiked into human serum samples. With the advantages of fast, easy operation and high-performance, this biosensing strategy might be an alternative tool for clinical diagnosis of HIV infection.

A one-step fluorescent biosensing strategy for highly sensitive detection of HIV-related DNA based on strand displacement amplification and Mg²⁺-dependent DNAzyme reaction.     

Label-free fluorescent aptasensor for chloramphenicol based on hybridization chain reaction amplification and G-quadruplex/N-methyl mesoporphyrin IX complexation

The use of the broad-spectrum antibiotic chloramphenicol (CAP) in food is strictly regulated or banned in many countries. Herein, for the sensitive, rapid, and specific detection of CAP in milk, a label-free fluorescence strategy was established based on guanine (G)-quadruplex/N-methyl mesoporphyrin IX (NMM) complex formation and hybridization chain reaction (HCR) amplification. In this system, CAP can specifically bind to an aptamer (Apt) to release an Apt-C sequence from double-stranded DNA (Apt·Apt-C). Apt-C, can further hybridize with a functional hairpin DNA probe to release a primer sequence. The released primer sequence causes HCR and the formation of a nicked double-helix polymer, which contains G-quadruplex DNA. The recognition of G-quadruplex DNA by the NMM fluorochrome results in fluorescence enhancement. Consequently, CAP can be quantitatively detected by measuring the fluorescence intensity at 612 nm. The reliability of the aptasensor method was confirmed by comparison with an enzyme-linked immunosorbent assay. The proposed aptasensor was found to have a limit of detection of 0.8 pg mL⁻¹ for CAP. Moreover, when the aptasensor was applied to the detection of CAP in milk samples, the average recoveries were 99.8–108.3% with relative standard deviations of 4.5–5.2%. Thus, this CAP detection method, which is rapid with high sensitivity and selectivity, has considerable potential for a wide range of food analysis applications.

For the sensitive and specific detection of CAP in milk, a label-free fluorescence strategy was established based on guanine (G)-quadruplex/N-methyl mesoporphyrin IX (NMM) complex formation and hybridization chain reaction (HCR) amplification.     

Comparison of detection and signal amplification methods for DNA microarrays

One of the factors limiting the use of DNA microarray technology for the detection of pathogenic organisms from clinical and environmental matrices has been inadequate assay sensitivity. To assess the effectiveness of post-hybridization secondary detection steps to enhance the sensitivity of DNA microarray-based pathogen detection, we evaluated a panel of 11 commercial and novel hybridization detection and signal amplification methods (direct labeling, indirect aminoallyl labeling, antibody, DNA dendrimers, viral particles, internally fluorescent nanoparticles, tyramide signal amplification, resonance light scattering nanoparticles and quantum dots) using a multiplex PCR and spotted long oligonucleotide microarray for Vibrio cholerae. Quantitative parameters such as sensitivity, signal intensity, background, assay complexity, time and cost were assessed and provide comparative criteria to be considered for DNA microarray experimental design. While the most important parameter is likely to vary based on the assay, when weighted equally, the findings suggest that recognition element- and dye-functionalized viral particles provide the most attractive option for microarray detection and signal amplification.     

An aptasensor for the label-free detection of thrombin based on turn-on fluorescent DNA-templated Cu/Ag nanoclusters

A highly sensitive thrombin aptasensor was constructed based on the alteration of the aptamer conformation induced by the target recognition and the turn-on fluorescence due to the proximity of two darkish DNA-templated copper/silver nanoclusters (DNA-Cu/Ag NCs). Two DNA templates were designed as the functional structures consisting of the Cu/Ag NC-nucleation segment located at two termini or one terminus and the aptamer segment in the middle of a DNA template. Two darkish DNA-Cu/Ag NCs came close to each other when the aptamer combined with the target due to the conformational alteration of the aptamer structure, resulting in an increased fluorescence signal readout. Thrombin was sensitively determined as low as 1.6 nM in the range of 1.6–8.0 nM with a high selectivity. Finally, this sensor succeeded in detecting thrombin in a real fetal bovine serum.

A highly sensitive thrombin aptasensor was constructed based on the alteration of the aptamer conformation induced by the target recognition and the turn-on fluorescence due to the proximity of two darkish DNA-templated copper/silver nanoclusters (DNA-Cu/Ag NCs).     

An ultrasensitive electrochemical sensing platform for the detection of cTnI based on aptamer recognition and signal amplification assisted by TdT

We have developed an ultrasensitive and highly specific electrochemical sensing platform for the detection of cardiac troponin I (cTnI), a recognized biomarker for the diagnosis of acute myocardial infarction (AMI) and related cardiovascular diseases (CVDs). This strategy is based on the assists of terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification and the specific recognition between cTnI and the aptamer of cTnI. In this experiment, we prepared a gold electrode that modified with probe 2 (P2), in the presence of cTnI, the aptamer of cTnI that in probe 1 (P1)/aptamer complexes bond with cTnI specifically and release the free P1. P1 would bind with P2, resulting in the formation of 3′-OH of DNA. In the presence of terminal deoxynucleotidyl transferase (TdT) and dTTP, TdT mediated P1 to extend and formed the structure of poly T. Methylene blue (MB)-poly A hybridized with the extended poly T and generated an electrochemical signal. The detection limit can be as low as 40 pg mL⁻¹. This sensor was also successfully applied to the detection of cTnI in numerous spiked biological samples, and it can be a great reference for the clinical diagnosis, prognosis, and treatment of CVDs and AMI.

We have developed an ultrasensitive and highly specific electrochemical sensing platform for the detection of cardiac troponin I (cTnI), a recognized biomarker for the diagnosis of acute myocardial infarction (AMI) and related cardiovascular diseases (CVDs).     

Symmetric exponential amplification reaction-based DNA nanomachine for the fluorescent detection of nucleic acids

By introducing palindromic sequences into the classical exponential amplification reaction (EXPAR), we constructed a new palindromic fragment-incorporated multifunctional hairpin probe (P-HP)-mediated symmetric exponential amplification reaction (S-EXPAR), to significantly reduce the background signal caused by inherent nonspecific amplification. A G-triplex/ThT complex was used as the signal reporter for the proposed label-free DNA nanomachine. The P-HP consists of five functional regions: a C-rich region (C), a target DNA recognition region (T′), two nicking sites (X′) and a palindromic fragment (P). When target DNA (T) hybridizes with P-HP, the palindromic fragment at the 3′ end of P-HP is fully exposed. Then, the P-HP/T duplexes hybridize with each other through the exposed P, and EXPAR occurs automatically and continuously on both sides of P under the synergistic effect of polymerase and nicking endonuclease. This is called the S-EXPAR assay. In this system, one T converts to a large number of G-triplex fragments, which can combine with ThT within a short time. The G-triplex/ThT complexes formed act as the signal reporter in a label-free and environmentally friendly format. In this way, the limit of detection of this method is as low as 10 pM with a dynamic response range of 10 pM to 300 nM. In addition, this method can detect other nucleic acids by simply changing the T′ region of the P-HP. Thus, the proposed DNA nanomachine is a potential alternative method for nucleic acid detection.

This label-free and ultra-low background signal DNA nanomachine was based on P-HP mediated S-EXPAR and the G-triplex/ThT complex.     

A sensitive platform for DNA detection based on organic electrochemical transistor and nucleic acid self-assembly signal amplification

Highly sensitive detection of DNA is of great importance for the detection of genetic damage and errors for the diagnosis of many diseases. Traditional highly sensitive organic electrochemical transistor (OECT)-based methods mainly rely on good conductivity materials, which may be limited by complex synthesis and modification steps. In this work, DNA biosensor based on OECT and hybridization chain reaction (HCR) signal amplification was demonstrated for the first time. Au nanoparticles were electrochemically deposited on the Au gate electrode to increase the surface area. Then, the HCR products, long negatively charged double-stranded DNA, were connected to the target by hybridization, which can increase the effective gate voltage offset of OECT. This sensor exhibited high sensitivity and even 0.1 pM target DNA could be directly detected with a significant voltage shift. In addition, it could discriminate target DNA from the mismatched DNA with good selectivity. This proposed method based on HCR in DNA detection exhibited an efficient amplification performance on OECT, which provided new opportunities for highly sensitive and selective detection of DNA.

A new method has been developed for DNA detection by integrating hybridization chain reaction signal amplification with organic electrochemical transistor device for the first time.     

An ultrasensitive fluorescent aptasensor for detection of cancer marker proteins based on graphene oxide–ssDNA

A novel biosensing platform was developed by integrating a new ssDNA aptamer and graphene oxide (GO) for highly sensitive and selective detection of liver cancer biomarkers (alpha-fetoprotein, AFP). The key concept of this biosensing platform is that the fluorescence of dye-modified ssDNA can be effectively quenched by GO after forming the hybrid structure of graphene oxide–ssDNA (GO–ssDNA). The AFP can selectively react with GO–ssDNA and lead to the decomposition of GO–ssDNA, which results in the recovery of fluorescence, and an increase in fluorescence intensity with the increasing concentration of AFP in the range of 0 to 300 pg mL⁻¹. The linear range was obtained from 1 to 150 pg mL⁻¹ and the detection limit was 0.909 pg mL⁻¹. Moreover, this biosensing platform can be applied to serum and cell imaging for the detection of AFP. The results show that the proposed biosensor has great potential application in AFP-related clinical diagnosis and research.

A novel biosensing platform was developed by integrating a new ssDNA aptamer and graphene oxide (GO) for highly sensitive and selective detection of liver cancer biomarkers (AFP).     

Dual signal amplification for microRNA-21 detection based on duplex-specific nuclease and invertase

MicroRNA-21 (miRNA-21) is a significant biomarker which is closely related to some kinds of diseases, such as cancer, cardiovascular disease and kidney disease. Therefore, the detection of miRNA-21 is of great importance and can provide essential information for disease diagnosis. In this study, we report a facile, sensitive assay for miRNA-21 detection using personal glucose meters (PGM). Biotinylated DNA strand linked invertase (Inv) is conjugated on the surface of streptavidin-coated magnetic beads (MBs) to form a MBs–DNA–Inv complex. Target miRNA-21 in the detection system is captured by the MBs–DNA–Inv probe through DNA/RNA hybridization. The duplex-specific nuclease (DSN) enzyme specifically cleaves the DNA to recycle the target miRNA and release invertase, thereby triggering the dual signal amplification and ensuring high sensitivity. Besides, we establish a linear relationship between PGM and different concentrations of miRNA-21 in the range of 10 to 200 pM. The limit of detection is 1.8 pM, which is more sensitive than some of the previous reports. In addition, the biosensor exhibits excellent sequence selectivity and single-base mutation can be discriminated. Moreover, the expression of miRNA-21 is confirmed in urine from mice by our method, which is in good accordance with the qRT-PCR result. Therefore, a dependable, low-cost strategy for the detection of miRNA has been established and it meets the latest analytical demands for miRNA determination that is suitable for the public.

A portable and sensitive method based on duplex-specific nuclease and MBs–DNA–Inv conjugate has been developed for miRNA-21 detection by using a personal glucose meter as the signal collector.     

The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157:H7

Escherichia coli O157:H7 (E. coli O157:H7) is a common foodborne morbigenous microorganism, which can spread through fecal-oral transmission. Humans can be infected by ingesting foods and water contaminated with E. coli O157:H7, which can cause various symptoms. In present study, we have successfully developed a quick and hypersensitive fluorescent probe-based Recombinase-aided amplification (RAA) method and applied in E. coli O157:H7 detection at 39 °C in 20 min. The sensitivity of the assay in pure E. coli O157:H7 suspension was 5.6 × 10⁰ CFU/mL. The fluorescent probe-based RAA assay was further applied in three samples, and the limit of detection (LOD) in skimmed milk, lettuces and lake water was 5.4 × 10¹ CFU/mL, 7.9 × 10¹ CFU/mL and 5.2 × 10¹ CFU/mL, separately. This method showed a high sensitivity and short detection time, which has the feasible application in on-site test in real samples.