Single Particle Inductively Coupled Plasma Mass Spectrometry-Based Homogeneous Detection of HBV DNA with Rolling Circle Amplification-Induced Gold Nanoparticle Agglomeration.

A highly sensitive and simple method based on rolling circle amplification (RCA) and single particle inductively coupled plasma mass spectrometry (spICP-MS) was proposed for the homogeneous detection of hepatitis B virus (HBV) deoxyribonucleic acid (DNA). In the presence of target DNA, long ssDNA possessing a large number of repeating sequence units was generated by RCA. DNA-labeled AuNP probes assembled into long chains based on complementary base pairing, further aggregating into large particles. Small Au NPs hardly produced pulse signals in spICP-MS; obvious pulse signals appeared in spICP-MS after the agglomeration of Au NPs caused by the addition of RCA products and spermidine. On the basis of this, the homogeneous detection of target DNA was realized by spICP-MS with high sensitivity. Under optimal conditions, the proposed method exhibited a good linear relationship between the frequency of the pulse signal of Au in spICP-MS and the concentration of target HBV DNA in the range of 10-2000 fmol L-1 (R = 0.997), the limit of detection was 5.1 fmol L-1, and the relative standard deviation was 3.7-6.8%. Recoveries of 94.2-108% were obtained for target DNA in spiked serum samples, demonstrating a good matrix tolerance ability for the method.

Elemental Mass Spectrometry and Fluorescence Dual-Mode Strategy for Ultrasensitive Label-Free Detection of HBV DNA.

This work reported a simple and ultrasensitive label-free method for the detection of hepatitis B virus (HBV) DNA by combining hyperbranched rolling circle amplification (HRCA) with dual-mode detection by inductively coupled plasma mass spectrometry (ICP-MS) and fluorescence using ruthenium complex [Ru(bpy)2dppz]2+ (bpy = 2,2'-bipyridine, dppz = dipyrido [3,2-a:2',3'-c] phenazine) as a dual functional probe. An HBV DNA-initiated HRCA system was designed to realize the highly efficient amplification of HBV DNA with the generation of a mass of dsDNA. Also, the [Ru(bpy)2dppz]2+ probe was then added to intercalate into the dsDNA products, resulting in strong fluorescence recovery of the probe for fluorescence detection. Meanwhile, using a biotin-modified primer in HRCA, the dsDNA-[Ru(bpy)2dppz]2+ complexes could be captured by the avidin-coated 96-well plates, and the captured [Ru(bpy)2dppz]2+ probe was later desorbed by acid for ICP-MS detection. The linear range of the proposed method was 3.5-200 amol L-1 and the limit of detection (LOD) was 1 amol L-1 for ICP-MS detection, while the linear range was 20-500 amol L-1 and the LOD was 9.6 amol L-1 for fluorescence detection. The developed method was applied to human serum sample analysis, and the analytical results coincided very well with those obtained by the real-time polymerase chain reaction (PCR) method. The developed dual-mode label-free detection method was ultrasensitive, simple, and accurate, showing great potential for therapeutic monitoring of HBV infection.

MNAzyme-Catalyzed Amplification Assay with Lanthanide Tags for the Simultaneous Detection of Multiple microRNAs by Inductively Coupled Plasma-Mass Spectrometry.

Quantification of multiple disease-related microRNAs (miRNAs) is of great significance for clinical diagnosis. Based on the simultaneous multiple element detection ability of inductively coupled plasma-mass spectrometry (ICP-MS) and good specificity of multicomponent nucleic acid enzymes (MNAzymes), a novel and simple method based on the MNAzyme amplification strategy and lanthanide labeling coupled with ICP-MS detection was proposed for the sensitive and simultaneous detection of three miRNAs (miRNA-21, miRNA-155, and miRNA-10b). Specifically, a probe consisting of streptavidin-modified magnetic beads (SA-MBs) and three DNA substrates labeled with lanthanide tags (159Tb/165Ho/175Lu) was constructed. In the presence of target miRNAs, three pairs of MNAzymes were assembled where each pair was hybridized with the corresponding miRNA, and then the substrates on the SA-MBs were cleaved by the activated MNAzymes, continuously releasing the fragment with lanthanide tags. The released lanthanide tags in the supernatant were collected after magnetic separation and analyzed by ICP-MS, realizing the simultaneous quantification of multiple miRNAs. The correlation of the lanthanide tag signal with the miRNA concentration fitted well in a linear model in the range of 50-1000 pmol L-1 (miRNA-21) and 50-2000 pmol L-1 (miRNA-155 and miRNA-10b). The limits of detection for three miRNAs were 11-20 pmol L-1, with the relative standard deviations of 2.2-2.7%. The recoveries of target miRNAs in the human serum and HepG-2 cells were in the range of 87.2-111% and 93.3-111%, respectively. Overall, the method is ideal for the simultaneous quantification of multiple miRNAs with advantages of low spectral interference, high sensitivity, good selectivity, and strong resistance to the complex matrix.

Dual-mode detection of avian influenza virions (H9N2) by ICP-MS and fluorescence after quantum dot labeling with immuno-rolling circle amplification.

Avian influenza virus (AIVs), hosted in poultry, are the pathogens of many poultry diseases and human infections, which bring huge losses to the poultry breeding industry and huge panic to society. Therefore, it is of great significance to establish accurate and sensitive detection methods for AIVs. In this work, a dual-mode detection method based on immuno-rolling circle amplification (immuno-RCA) and quantum dots (QDs) labeling for inductively coupled plasma mass spectrometry (ICP-MS) and fluorescence detection of H9N2 AIV was developed. The dual-mode detection of the QDs by ICP-MS and fluorescence is used to achieve mutual verification within the analysis results, thus improving the accuracy of the method. With the immuno-RCA, the sensitivity of the method was increased by two orders of magnitude. The limit of detection of the proposed method is 17 ng L-1 and 61 ng L-1, and the linear range of the proposed method is 0.05-5 ng mL-1 and 0.1-5 ng mL-1 with ICP-MS and fluorescence detection, respectively. The relative standard deviation (n = 7) is 4.9% with ICP-MS detection and 3.1% with fluorescence detection. Furthermore, the proposed method was applied to the analysis of chicken serum samples, no significant different was found for two modes detection and the recoveries of the spiking experiments are acceptable, indicating that the method has good practical potential for real sample analysis.

A highly sensitive assay of DNA based on inductively coupled plasma mass spectrometry detection with gold nanoparticle amplification and isothermal circular strand-displacement polymerization reaction.

In this work, a simple, sensitive and specific assay for DNA was proposed by combining inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticle (AuNPs) amplification and isothermal circular strand-displacement polymerization reaction (ICSDPR). First, AuNPs were decorated with hairpin-structured DNA (HP-DNA) through Au-S bond to form the AuNPs probe. The ICSDPR was conducted on AuNPs probe in a homogeneous phase to realize the dual amplification and simplify the analytical process at the same time. By using a biotin modified primer, AuNPs were connected with biotins after the ICSDPR, then captured by the streptavidin modified magnetic beads (SA-MBs), and finally detected by ICP-MS. Many key factors including probe volume, hybridization time, polymerase amount, primer concentration, enzyme reaction time, SA-MBs capture time and desorption time were optimized. Under the optimized condition, the proposed method could detect target DNA as low as 45 zmol (8.9 fM in 5 µL) in a relative short time (about 4.5 h) with good specificity, and the linear range of this method is 0.1-10000 pM, the relative standard deviations are in the range of 3.6-6.4%. The proposed method was applied for determination of target DNA in human serum samples, the recovery for the spiked human serum samples is in the range of 84-120%. It demonstrates a good application potential of the developed method for biological studies and clinical diagnosis of human pathogenic diseases.

Lectin affinity based elemental labeling with hybridization chain reaction for the sensitive determination of avian influenza A (H9N2) virions.

Avian influenza virus (AIV) as a type of highly pathogenic influenza A virus, can not only cause serious illness and death in poultry but also threat human health and lead to public panic. Rapid, sensitive detection of AIV is urgent and significant for prevention and timely control of influenza epidemics. Herein, we reported an inductively coupled plasma mass spectrometry (ICP-MS) based method for the analysis of AIV virions on the basis of the selective recognition between lectin Con A and glycoproteins on AIV surface and signal amplification of hybridization chain reaction (HCR). With H9N2 as the model AIV, the limit of detection was down to 0.12 ng mL-1 due to the dual amplification effect of AuNPs and HCR, and the linear range was 0.4-50 ng mL-1 with the relative standard deviation of 7.9% for seven replicate detections of 2 ng mL-1 H9N2 virions. Furthermore, the applicability of the method for the analysis of real biological samples was demonstrated by the spiking tests. The proposed approach is highly specific and sensitive for the detection of AIV with good application potential in early diagnosis, which is helpful for the prevention of influenza outbreak.

Gold nanoparticle labeling with tyramide signal amplification for highly sensitive detection of alpha fetoprotein in human serum by ICP-MS.

In this work, we developed an immunoassay based on tyramide signal amplification (TSA) and gold nanoparticles (Au NPs) labeling for highly sensitive detection of alpha fetoprotein (AFP) by inductively coupled plasma mass spectrometry (ICP-MS). AFP was captured by anti-AFP1 coating on the 96-well plate and labeled by anti-AFP2-horseradish peroxidase (HRP), in which the HRP can catalyze the deposition of biotinylated tyramine on the nearby protein. Then the streptavidin (SA)-Au NPs was labeled on the deposited biotinylated tyramine as the intensive signal probe for ICP-MS measurement. Under the optimal experimental conditions, the limit of detection of the developed method for AFP was 1.85pg/mL and the linear range was 0.005-2ng/mL. The relative standard deviation for seven replicate detections of 0.01ng/mL AFP was 5.2%. The proposed method was successfully applied to the detection of AFP in human serum with good recoveries. This strategy is highly sensitive and easy to operate, and can be extended to the sensitive detection of other biomolecules in human serum.

Simultaneous detection of MCF-7 and HepG2 cells in blood by ICP-MS with gold nanoparticles and quantum dots as elemental tags.

In this work, we demonstrate a novel method based on inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticles (Au NPs) and quantum dots (QDs) labeling for the simultaneous counting of two circulating tumor cell lines (MCF-7 and HepG2 cells) in human blood. MCF-7 and HepG2 cells were captured by magnetic beads coupled with anti-EpCAM and then specifically labeled by CdSe QDs-anti-ASGPR and Au NPs-anti-MUC1, respectively, which were used as signal probes for ICP-MS measurement. Under the optimal experimental conditions, the limits of detection of 50 MCF-7, 89 HepG2 cells and the linear ranges of 200-40000 MCF-7, 300-30000 HepG2 cells were obtained, and the relative standard deviations for seven replicate detections of 800 MCF-7 and HepG2 cells were 4.6% and 5.7%, respectively. This method has the advantages of high sensitivity, low sample consumption, wide linear range and can be extended to the simultaneous detection of multiple CTC lines in human peripheral blood.