Real-time wash-free detection of unlabeled PNA-DNA hybridization using discrete FET sensor.

We demonstrate an electrochemical sensor for detection of unlabeled single-stranded DNA using peptide nucleic acid (PNA) probes coupled to the field-effect transistor (FET) gate. The label-free detection relies on the intrinsic charge of the DNA backbone. Similar detection schemes have mainly concentrated on sensitivity improvement with an emphasis on new sensor structures. Our approach focuses on using an extended-gate that separates the FET and the sensing electrode yielding a simple and mass fabricable device. We used PNA probes for efficient hybridization in low salt conditions that is required to avoid the counter ion screening. As a result, significant part of the target DNA lies within the screening length of the sensor. With this, we achieved a wash-free detection where  typical gate potential shifts are more than 70 mV with 1 µM target DNA. We routinely obtained a real-time, label- and wash-free specific detection of target DNA in nanomolar concentration with low-cost electronics and the responses were achieved within minutes after introducing targets to the solution. Furthermore, the results suggest that the sensor performance is limited by specificity rather than by sensitivity and using low-cost electronics does not limit the sensor performance in the presented sensor configuration.

Integrated Acoustic Separation, Enrichment, and Microchip Polymerase Chain Reaction Detection of Bacteria from Blood for Rapid Sepsis Diagnostics.

This paper describes an integrated microsystem for rapid separation, enrichment, and detection of bacteria from blood, addressing the unmet clinical need for rapid sepsis diagnostics. The blood sample is first processed in an acoustophoresis chip, where red blood cells are focused to the center of the channel by an acoustic standing wave and sequentially removed. The bacteria-containing plasma proceeds to a glass capillary with a localized acoustic standing wave field where the bacteria are trapped onto suspended polystyrene particles. The trapped bacteria are subsequently washed while held in the acoustic trap and released into a polymer microchip containing dried polymerase chain reaction (PCR) reagents, followed by thermocycling for target sequence amplification. The entire process is completed in less than 2 h. Testing with Pseudomonas putida spiked into whole blood revealed a detection limit of 1000 bacteria/mL for this first-generation analysis system. In samples from septic patients, the system was able to detect Escherichia coli in half of the cases identified by blood culture. This indicates that the current system detects bacteria in patient samples in the upper part of the of clinically relevant bacteria concentration range and that a further developed acoustic sample preparation system may open the door for a new and faster automated method to diagnose sepsis.

Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes.

Genotyping in closed tube is commonly performed using polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide probes using fluorescence resonance energy transfer (FRET). Here we introduce a homogeneous human leukocyte antigen (HLA)-DQA1∗05 end-point PCR assay based on switchable lanthanide luminescence probe technology and a simple dried blood sample preparation. The switchable probe technology is based on two non-luminescent oligonucleotide probes: one carrying a non-luminescent lanthanide chelate and the other carrying a light-absorbing antenna ligand. Hybridization of the probes in adjacent positions to the target DNA leads to the formation of a highly luminescent lanthanide chelate complex by self-assembly of the reporter molecules. Performance of the HLA-DQA1∗05 assay was evaluated by testing blood samples collected on sample collection cards and was prepared by lysing the punched samples (3-mm discs) using alkaline reaction conditions and high temperature. Testing of 147 blood samples yielded 100% correlation to the heterogeneous DELFIA technology-based reference assay. Genotyping requires carefully designed probe sequences able to discriminate matched and mismatched target sequences by hybridization. Furthermore, definite genotype discrimination was achieved because inherently non-luminescent switchable probes together with time-resolved measurement mode led to very low background signal level and, therefore, very high signal differences averaging 54-fold between DQA1∗05 and other alleles.

Lanthanide chelate complementation and hydrolysis enhanced luminescent chelate in real-time reverse transcription polymerase chain reaction assays for KLK3 transcripts.

The requirement for high-performance reporter probes in real-time detection of polymerase chain reaction (PCR) has led to the use of time-resolved fluorometry of lanthanide chelates. The aim of this study was to investigate the applicability of the principle of lanthanide chelate complementation (LCC) in comparison with a method based on hydrolysis enhancement and quenching of intact probes. A real-time reverse transcription (RT) PCR assay for kallikrein-related peptidase 3 (KLK3, model analyte) was developed by using the LCC detection method. Both detection methods were tested with a standard series of purified PCR products, 20 prostatic tissues, 20 healthy and prostate cancer patient blood samples, and female blood samples spiked with LNCaP cells. The same limit of detection was obtained with both methods, and two cycles earlier detection with the LCC method was observed. KLK3 messenger RNA (mRNA) was detected in all tissue samples and in 1 of 20 blood samples identically with both methods. The background was 30 times lower, and the signal-to-background (S/B) ratio was 3 times higher, when compared with the reference method. Use of the new reporter method provided similar sensitivity and specificity as the reference method. The lower background, the improved S/B ratio, and the possibility of melting curve analysis and single nucleotide polymorphism (SNP) detection could be advantages for this new reporter probe.

Homogeneous duplex polymerase chain reaction assay using switchable lanthanide fluorescence probes.

We have developed a duplex polymerase chain reaction (PCR) assay based on switchable lanthanide chelate complementation probes. In the complementation probe technology, two nonfluorescent oligonucleotide probes, one labeled with a lanthanide ion carrier chelate and another with a light absorbing antenna ligand, form a fluorescent complex by self-assembly of the reporter molecules when the two probes are hybridized in adjacent positions to the target DNA. Here we report the synthesis of a new terbium(III) (Tb(III)) ion carrier chelate and a new light-absorbing antenna ligand for Tb(III) and the development of a duplex Chlamydia trachomatis (Ct) PCR assay. For the detection of Ct in urine samples, a specific sequence in Ct cryptic plasmid was amplified and detected using europium(III) (Eu(III)) complementation probes. An internal amplification control was amplified in each reaction and detected using Tb(III) complementation probes to verify the Ct negative results. Ct bacteria were concentrated from urine samples with a rapid and simple centrifugation-based sample preparation method. Good diagnostic accuracy (99-100%) was achieved, and also Ct positive reactions yielded a very high Eu(III) signal-to-background ratio (maximum of 244). High performance of the complementation probes is advantageous when sample may contain impurities after a simple sample preparation.

Homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes.

We have developed a rapid and reliable bacteriophage quantification method based on measurement of phage single-stranded DNA (ssDNA) using switchable lanthanide chelate complementation probes. One oligonucleotide probe contains a non-fluorescent lanthanide ion carrier chelate and another probe is labeled with a light absorbing antenna ligand. Hybridization of the non-fluorescent complementation probes in adjacent positions on the released bacteriophage ssDNA leads to high local concentrations of the lanthanide ion carrier chelate and the antenna ligand, inducing formation of a fluorescent lanthanide chelate complex. This method enables monitoring of bacteriophage titers in a 20 min assay with a dynamic range of 10(9)-10(12) cfu/mL in a microtiter well format. While designed for titering filamentous bacteriophage used in phage display, our method also could be implemented in virological research as a tool to analyze ssDNA virus reproduction.

High-performance closed-tube PCR based on switchable luminescence probes.

We introduce a switchable lanthanide luminescence reporter technology based closed-tube PCR for the detection of specific target DNA sequence. In the switchable lanthanide chelate complementation based reporter technology hybridization of two nonfluorescent oligonucleotide probes to the adjacent positions of the complementary strand leads to the formation of a highly fluorescent lanthanide chelate complex. The complex is self-assembled from a nonfluorescent lanthanide chelate and a light-harvesting antenna ligand when the reporter molecules are brought into close proximity by the oligonucleotide probes. Outstanding signal-to-background discrimination in real-time PCR assay was achieved due to the very low background fluorescence level and high specific signal generation. High sensitivity of the reporter technology allows the detection of a lower concentration of amplified DNA in the real-time PCR, resulting in detection of the target at the earlier amplification cycle compared to commonly used methods.

Rapid homogeneous PCR assay for the detection of Chlamydia trachomatis in urine samples.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays.