We describe a scheme for biomolecule enumeration by converting nanometer-scale specific molecular recognition events mediated by rolling-circle amplification to fluorescent micrometer-sized DNA molecules amenable to discrete optical detection. Our amplified single-molecule detection (SMD) approach preserves the discrete nature of the molecular population, allowing multiplex detection and highly precise quantification of molecules over a dynamic range of seven orders of magnitude. We apply the method for sensitive detection and quantification of the bacterial pathogen Vibrio cholerae.

In this letter, we demonstrate a new principle for diagnostics based on DNA sequence detection using single-stranded oligonucleotide tagged magnetic nanobeads. The target DNA is recognized and volume-amplified to large coils by circularization of linear padlock probes through probe hybridization and ligation, followed by rolling circle amplification (RCA). Upon hybridization of the nanobeads in the RCA coils, the complex magnetization spectrum of the beads changes dramatically, induced by the attached volume-amplified target molecules. We show that the magnetization spectrum of the nanobeads can be used for concentration determination of RCA coils down to the pM range, thus creating the opportunity for nonfluorescence-based cost-efficient high-sensitivity diagnostics tool. We also show that the bead incorporation in the coils is diffusion-controlled and consequently may be accelerated by incubating the sample at higher temperatures.

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To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection. Overprescription and extensive use of antibiotics have selected for resistant bacteria at an alarmingly rapid rate, and we are now facing one of the greatest medical challenges of our time (1). Today, both the diagnosis of bacterial infections and determination of antibiotic susceptibility profiles (ASP) are slow and tedious processes. As a consequence, a patient might be given an antibiotic that has no effect on infections caused by resistant bacteria. Thus, there is a considerable need for new techniques enabling quick and specific diagnosis along with characterization of an ASP in order to guide correct treatment, reduce the use of broad-spectrum antibiotics, and slow the development of resistance.In the last few decades, we have seen an amazing development of novel molecular methods to detect bacterial pathogens and their resistance genes and resistance mutations (2-5). These new hybridization/PCR-based methods are generally faster and more sensitive than are the classical phenotypic methods, but they also suffer from serious drawbacks that have often reduced their general use. An intrinsic limitation of all genotypic methods that identify resistance mutations or genes is that they detect only the potential for resistance (i.e., presence of a resistance gene/mutation), whereas phenotypic methods detect the realization of susceptibility (i.e., no growth in the presence of antibiotic). For a clinician, the realization of susceptibility measure is far more relevant as a basis for a therapeutic decision.Padlock probes are oligonucleotides with target-specific ends, which upon perfect target recognition can be enzymatically joined (6). Reacted probes can be amplified by rolling circle amplification (RCA). RCA is a linear amplification technique for the replication of DNA circles, such as reacted padlock probes, and the product is a single-stranded DNA concatemer containing around 1,000 copies of a 100-mer t...

The possibility for conducting multiplex detection of DNA-sequences using the volume-amplified magnetic nanobead detection assay [Stromberg, M.; Goransson, J.; Gunnarsson, K.; Nilsson, M.; Svedlindh, P. and Strømme, M. Nano Lett. 2008 , 8, 816-821] was investigated. In this methodology, a batch consisting of a mixture of several sizes of probe-tagged magnetic beads was used for detection of several types of targets in the same compartment. Furthermore, a nonlinear least-squares deconvolution procedure of the composite imaginary part of complex magnetization vs frequency spectra based on the Cole-Cole model was applied to analyze the data. The results of a quantitative biplex analysis experiment were compared with the corresponding separate single-target assays. Finally, triplex analysis was briefly demonstrated qualitatively. Biplex and triplex detection were found to perform well qualitatively. Biplex detection was found to enable a rough target quantification. Multiplex detection may become a complement to performing multiple separate single-target assays for, e.g., parallel detection of multiple infectious pathogens. Multiplex detection also permits robust relative quantification and inclusion of an internal control to improve quantification accuracy.