We developed an integrated chip for real-time amplification and detection of nucleic acid using pH-sensing complementary metal-oxide semiconductor (CMOS) technology. Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during nucleotide incorporation rather than relying on indirect measurements such as fluorescent dyes. This is a label-free, non-optical, real-time method for detecting and quantifying target sequences by monitoring pH signatures of native amplification chemistries. The chip has ion-sensitive field effect transistor (ISFET) sensors, temperature sensors, resistive heating, signal processing and control circuitry all integrated to create a full system-on-chip platform. We evaluated the platform using two amplification strategies: PCR and isothermal amplification. Using this platform, we genotyped and discriminated unique single-nucleotide polymorphism (SNP) variants of the cytochrome P450 family from crude human saliva. We anticipate this semiconductor technology will enable the creation of devices for cost-effective, portable and scalable real-time nucleic acid analysis.

Point-of-care diagnostics for detection of genetic sequences require biosensing platforms that are sensitive to the target sequence, and are also fast, mass-manufacturable, and -ideally -disposable. Conventional lab-based methods of detecting DNA sequences rely on optical methods, typically by the addition of fluorescent tags to the target DNA that in turn latches onto a DNA probe sequence only if there is a match between the two. These techniques are cumbersome as they require upfront tagging of the DNA with expensive reagents and laboratory equipment to detect the optical signals. Recently, developments have been made in transferring these optical methods to inexpensive CMOS ICs [1], although the requirement for tagging remains. Magnetic beads offer an alternative means of tagging the DNA and their presence can be detected by the shift in resonant frequency of an on-chip LC tank [2]. There have also been attempts based on "label-free" electrochemical detection using FETs [3,4], but none of these have been implemented in unmodified standard CMOS.We have developed a pioneering all-electrical approach that does not require tagging of DNA. The principle of label-free electrochemical DNA detection using an ion-sensitive field effect transistor (ISFET) was originally described in [5]. However, this prior art used a custom (non-CMOS) FET with an exposed Al 2 O 3 gate insulator, a macroscopic reference electrode, and a heat bath with control unit to control the temperature of the reagents; furthermore, it was only able to detect a single DNA sequence, which has limited application. The SOC for DNA detection presented herein integrates the discrete laboratory apparatus of [5] into a single IC while using standard CMOS manufacturing (unlike the all-electrical approach in [6]), and moreover has scalability to the detection of multiple DNA sequences by use of multiple on-chip ISFET sensors, temperature sensors and heaters along with ADCs and digital IO. This single-chip solution can be incorporated into disposable cartridges that are interchangeable depending on the DNA sequences to be detected. The structure of the cartridge is shown in Fig. 27.4.1 and consists of the IC embedded within a microfluidic assembly in which there are microchambers with access channels for the delivery of reagents. The ISFETs are implemented as unmodified floating-gate pMOS devices [7].When there is a match between target DNA from a sample and the on-chip DNA probes, H + ions are released, changing the pH of the reagents inside the microchamber in which the match took place (Fig. 27.4.1). ISFETs are sensitive to the pH of liquids on the passivation interface above their floating gates and this manifests itself as a shift in the V t of the ISFET where V t is the threshold voltage referred to a reference electrode that sets the potential of the electrolyte [8]. Hence, the IC can detect whether a reaction has taken place in any of the chambers by monitoring the V t of the ISFETs beneath those chambers. There are many ways to do this, one of whic...